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Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta

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PLOS Pathogens
Oct 2017



MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript.

Keywords: Rice stripe virus (水稻条纹病毒), NS3 (NS3), Double-stranded RNA binding protein (双链RNA结合蛋白), Primary-miRNA (初级miRNA), miRNA (miRNA), Plant-microbe interaction (植物 - 微生物交互作用)


MicroRNAs (miRNAs) are processed from their primary transcripts (pri-miRNAs) by the RNase III enzyme DICER-LIKE 1 (DCL1) with the help of the double-stranded RNA (dsRNA) binding protein HYPONASTIC LEAVES1 (DRB1/HYL1) and the zinc finger protein SERRATE (SE). Rice stripe virus (RSV) infection broadly perturbs miRNA accumulation. We found that RSV-encoding nonstructural protein 3 (NS3) promotes miRNA accumulation by downregulating pri-miRNAs through interaction with DRB1 in rice (Zheng et al., 2017). To reveal how NS3 enhances pri-miRNA processing, we used co-immunoprecipitation (Co-IP) to illustrate the relationship of NS3, DRB1 and pri-miRNA in vivo. This protocol contributes to understand association patterns between two proteins and one RNA transcript.

Materials and Reagents

  1. Pipette (RNase free 1 ml, 0.2 ml and 0.02 ml, Axygen)
  2. Miracloth (Merck, Calbiochem, catalog number: 475855 )
  3. Centrifuge tube (1.5 ml, 2 ml and 50 ml) (Corning)
  4. 4- to 6- weeks old Nicotiana benthamiana (leaves, grow in green house)
  5. Agrobacterium tumefaciens (EHA105 strain, preserved in our lab)
  6. Expression plasmids: pEarleyGate202-DRB1, -mutant DRB1, pEarleyGate203-NS3, -mutant NS3 and pCAMBIA-artificial primary miRNA transcript, -mutant primary miRNA transcript (Constructed by ourselves)
  7. Double-distilled or MilliQ water (ddH2O)
  8. Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  9. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 70011069 )
  10. Glycine (Sigma-Aldrich, catalog number: V900144 )
  11. Liquid nitrogen
  12. Anti-Myc (9E10) monoclonal antibodies (Sigma-Aldrich, catalog numbers: M4439 ) and mouse IgG control (Thermo Fisher Scientific, Invitrogen, catalog number: 02-6502 )
  13. Protein G-agarose (Roche Diagnostics, catalog number: 11243233001 )
  14. Trizol
  15. Chloroform (Sigma-Aldrich, catalog number: 613304 )
  16. GlycoBlueTM coprecipitant (15 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9516 )
  17. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  18. Ethanol (Aladdin, catalog number: E111963 )
  19. DNase I (Promega, catalog number: M6101 )
  20. Sucrose (Sigma-Aldrich, catalog number: V900116 )
  21. Ficoll 400 (Sigma-Aldrich, catalog number: F9378 )
  22. Dextran T40 (Sigma-Aldrich, catalog number: 1179708 )
  23. HEPES (Sigma-Aldrich, catalog number: RDD002 )
  24. KOH
  25. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  26. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  27. EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 05892953001 )
  28. DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: 43817 )
  29. Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
  30. NP-40 (Sigma Aldrich, catalog number: NP40S )
  31. RNase inhibitor (RNaseOUT) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
  32. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: V900312 )
  33. Diethyl pyrocarbonate (Sigma-Aldrich, catalog number: 40718 )
  34. Honda buffer (see Recipes)
  35. High salt nuclear lysis buffer (see Recipes)
  36. Dilution buffer (see Recipes)
  37. IP buffer (see Recipes)


  1. Eppendorf pipettes suite (1 ml, 0.2 ml, 0.02 ml, 0.01 ml and 0.0025 ml)
  2. Thermomixer C (Eppendorf, model: Thermomixer® C , catalog number: 5382000023)
  3. Vacuum pump (FJC, model: 6912 )
  4. Rotator (Glas-Col, model: 099A MR1512 )
  5. Centrifuge (Eppendorf, models: 5424 R and 5804 R )
  6. Pico Ultrasonicator (Diagenode, model: Bioruptor® Pico, model: 4486126 )
  7. Vortex (Kylin-Bell Lab Instruments, model: VORTEX-5 )
  8. Pestle


  1. Material preparation
    Agrobacterium tumefaciens-mediated transient co-expression of corresponding proteins and RNAs was performed with 4- to 6-week old N. benthamiana. N. benthamiana leaves (~8 g). Harvest the samples at 3 dpi (days post inoculation).

  2. Nuclei isolation
    1. Cross-link N. benthamiana leaves with 1% formaldehyde in 1x PBS for 15 min.
      Note: Make sure the sample is submerged in the buffer, apply the vacuum for 5 min, release, Reapply vacuum, repeat this for 10 times.
    2. Add glycine to a final concentration of 0.125 M, mix well the buffer and apply vacuum for 5 min to stop cross-linking.
    3. Rinse the plants three times with sterile water. Remove excess water, freeze the plants in liquid nitrogen. If not proceed to the next step immediately, store the frozen plants at -80 °C.
    4. Grind the plant material into fine powder with liquid nitrogen in mortar. Transfer the power to a 50 ml centrifuge tube.
    5. For each sample (2 g), add 15 ml Honda buffer, rotate at 4 °C for 5 min.
    6. Filter through one layer of Miracloth, then wash the Miracloth by adding another 10 ml of Honda buffer (in a 50 ml centrifuge tube).
    7. Centrifuge at 3,500 x g for 5 min at 4 °C. Discard the supernatant, softly resuspend the pellet in 1 ml Honda buffer with 1 ml pipet with end-cut tip.
    8. Centrifuge at 3,500 x g for 5 min, wash the pellet once more with 1 ml Honda buffer. Centrifuge at 10,000 x g for 1 min and remove the supernatant thoroughly.

  3. Immunoprecipitation
    1. Add 2.5 volumes/weight high salt nuclear lysis buffer in nuclear pellets, resuspend nuclei and sonicate (30 sec long pulses, 30 sec intervals, 10 cycles) by using a Diagenode Bioruptor.
    2. Centrifuge (16,000 x g, 4 °C) for 10 min, take supernatant (400 μl) into a 2 ml centrifuge tube, add four volumes of dilution buffer, mix well, take out 1/20, and keep it as input.
    3. Add anti-Myc (10 μg/2 ml) antibody, rotate at 4 °C for 2 h, then add 50 μl of Protein G and rotate for another 2 h.
    4. Centrifuge at 1,500 x g for 5 min at 4 °C. Remove supernatant carefully and add IP buffer. Wash beads with IP buffer for 3 times.
    5. Take out 1/10 of IP extract for Western blot detection.
    6. Centrifuge at 1,500 x g for 5 min at 4 °C, remove the supernatant thoroughly.

  4. RNA extraction and RT-PCR
    1. Add 1 ml Trizol, 65 °C for 10 min.
    2. Add 200 μl chloroform, vortex, incubate the tubes on ice for 5 min.
    3. Centrifuge at 16,000 x g for 5 min.
    4. Take 400 μl aqueous phase.
    5. Add 1 μl GlycoBlue and 40 μl sodium acetate, then add 1 ml anhydrous ethanol, incubate at -80 °C, 2 h.
    6. Centrifuge at 16,000 x g for 5 min, discard the supernatant.
    7. Suspend the RNA pellet in 89 μl of nuclease-free water.
    8. Add 10 μl 10x DNase I buffer and 1 μl DNase I, 37 °C for 10 min.
    9. Add 300 μl nuclease-free water then 400 μl chloroform, vortex.
    10. Centrifuge at 16,000 x g for 5 min.
    11. Take 400 μl aqueous phase.
    12. Add 1 μl GlycoBlue and 40 μl sodium acetate, then add 1 ml anhydrous ethanol, -80 °C, 2 h.
    13. Centrifuge at 16,000 x g for 5 min, discard the supernatant.
    14. Suspend the RNA pellet in 20 μl of nuclease-free water.
    15. Reverse transcript by SuperScriptIII (follow the manufacturer's instructions) with gene-specific primer, and detect RNAs by RT-PCR (melting temp.: 95 °C, annealing temp.: 60 °C, cycle numbers: 30 cycles).

Data analysis

Co-IP products were reversely transcribed using gene specific primer. Reverse transcripts were separated by electrophoresis on a 2% agarose gel. As shown in Figure 1, both OsDRB1a and mOsDRB1a interacted with apri-miR528, but neither of them recognized mapri-miR528; and NS3 and mNS3 were associated with OsDRB1a instead of mOsDRB1a. With the expression of NS3, both OsDRB1a and mOsDRB1a interacted with apri-miR528. However, with the expression of mNS3, only mOsDRB1a was associated with apri-miR528. These results indicate that NS3 may act as a scaffold to mediate the interaction between OsDRB1 and pri-miRNA.

Figure 1. NS3 acts as a scaffold between DRB1 and pri-miRNA. RT-PCR detection of coimmunoprecipitated and input products of transiently co-expressed protein (DRB1a or mDRB1a), pri-miRNA (aprimiR528 or mapri-miR528), and protein (empty vector, NS3, or mNS3) in N. benthamiana. For more information, see Zheng et al., 2017.


  1. Keep all the buffers RNase-free.
  2. If you have many samples, isolates nuclei one by one, and freeze the nuclear pellet in the liquid nitrogen.
  3. More detailed information on nuclei isolation and immunoprecipitation can be obtained from Saleh et al., 2008 and Terzi et al., 2009.


  1. Honda buffer
    0.44 M sucrose
    1.25% Ficoll
    2.5% Dextran T40
    20 mM HEPES-KOH, pH7.4
    0.5% Triton X-100
    10 mM MgCl2
    20 U/ml RNase inhibitor
    5 mM DTT (freshly added)
    1x Cocktail Roche cOmplete (freshly added)
  2. High salt nuclear lysis buffer
    20 mM Tris-HCl, pH = 7.5
    500 mM NaCl
    4 mM MgCl2
    20 U/ml RNase inhibitor (freshly added)
    0.2% NP-40 (add fresh)
    5 mM DTT (add fresh)
    1x Cocktail Roche cOmplete (freshly added)
  3. Dilution buffer
    20 mM Tris-HCl, pH = 7.5
    4 mM MgCl2
    0.2% NP-40 (freshly added)
    5 mM DTT (freshly added)
    20 U/ml RNase inhibitor (freshly added)
    1x Cocktail Roche cOmplete (freshly added)
  4. IP buffer
    20 mM Tris-HCl, pH = 7.5
    100 mM NaCl
    4 mM MgCl2
    0.2% NP-40 (add fresh)
    5 mM DTT (add fresh)
    20 U/ml RNase inhibitor (add fresh)
    1x Cocktail Roche cOmplete (freshly added)


This work was supported by grants to JG. W. and L. Y. from the National Natural Science Foundation of China (Nos. 31722045; 31772128; 31701757 and 31201491); the National Basic Research Program 973 (2014CB138400); and the Natural Science Foundation of Fujian Province of China, Outstanding Young Scientific Research Plan and Excellent Talent Plan in the New Century of Fujian Province (JA3091 and 2014J06011). This protocol was modified from Saleh et al., 2008 and Terzi et al., 2009. We declare no conflicts of interest or competing interests.


  1. Saleh, A., Alvarez-Venegas, R. and Avramova, Z. (2008). An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3(6): 1018-1025.
  2. Terzi, L. C. and Simpson, G. G. (2009). Arabidopsis RNA immunoprecipitation. Plant J 59(1): 163-168.
  3. Zheng, L., Zhang, C., Shi, C., Yang, Z., Wang, Y., Zhou, T., Sun, F., Wang, H., Zhao, S., Qin, Q., Qiao, R., Ding, Z., Wei, C., Xie, L., Wu, J. and Li, Y. (2017). Rice stripe virus NS3 protein regulates primary miRNA processing through association with the miRNA biogenesis factor OsDRB1 and facilitates virus infection in rice. PLoS Pathog 13(10): e1006662.


微小RNA(miRNA)在植物生长,发育和微生物感染反应中发挥重要作用。 双链RNA结合蛋白1(DRB1)有助于将初级miRNA转录物加工成成熟的miRNA。 最近,我们发现水稻条纹病毒(RSV)编码的NS3蛋白与DRB1相关并促进RSV感染期间的miRNA生物合成(Zheng等人,2017)。 RNA共免疫沉淀(RIP)方法被用于鉴定DRB1,NS3和miRNA转录物之间的关联模式。

【背景】在双链RNA(dsRNA)结合蛋白HYPONASTIC LEAVES1(DRB1 / HYL1)的帮助下,通过RNA酶III酶DICER-LIKE1(DCL1)从其初级转录物(pri-miRNA) 锌指蛋白SERRATE(SE)。 水稻条纹病毒(RSV)感染广泛地干扰miRNA积累。 我们发现RSV编码的非结构蛋白3(NS3)通过与水稻中的DRB1相互作用下调pri-miRNAs来促进miRNA积累(Zheng等人,2017)。 为了揭示NS3如何增强pri-miRNA的加工,我们使用免疫共沉淀(Co-IP)来说明NS3,DRB1和pri-miRNA体内的关系。 该协议有助于了解两种蛋白质和一种RNA转录本之间的关联模式。

关键字:水稻条纹病毒, NS3, 双链RNA结合蛋白, 初级miRNA, miRNA, 植物 - 微生物交互作用


  1. 移液管(不含RNase 1ml,0.2ml和0.02ml,Axygen)
  2. Miracloth(Merck,Calbiochem,目录号:475855)
  3. 离心管(1.5毫升,2毫升和50毫升)(康宁)
  4. 4至6周龄的本生烟草(烟草,在温室里种植)
  5. 根癌土壤杆菌(EHA105菌株,在我们实验室保存)
  6. 表达质粒:pEarleyGate202-DRB1, - 突变型DRB1,pEarleyGate203-NS3, - 突变型NS3和pCAMBIA- em>人工初级miRNA转录本,突变初级miRNA转录本(由我们自己构建)
  7. 双蒸或MilliQ水(ddH2O)
  8. 甲醛(Sigma-Aldrich,目录号:F8775)
  9. PBS(Thermo Fisher Scientific,Gibco TM,目录号:70011069)
  10. 甘氨酸(Sigma-Aldrich,目录号:V900144)
  11. 液氮
  12. 抗Myc(9E10)单克隆抗体(Sigma-Aldrich,目录号:M4439)和小鼠IgG对照(Thermo Fisher Scientific,Invitrogen,目录号:02-6502)
  13. 蛋白质G-琼脂糖(Roche Diagnostics,目录号:11243233001)
  14. Trizol
  15. 氯仿(Sigma-Aldrich,目录号:613304)
  16. GlycoBlue TM共沉淀剂(15mg / ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9516)。
  17. 乙醇(Sigma-Aldrich,目录号:E7023)
  18. 乙醇(阿拉丁,目录号:E111963)
  19. DNase I(Promega,目录号:M6101)
  20. 蔗糖(Sigma-Aldrich,目录号:V900116)
  21. Ficoll 400(Sigma-Aldrich,目录号:F9378)
  22. 葡聚糖T40(Sigma-Aldrich,目录号:1179708)
  23. HEPES(Sigma-Aldrich,目录号:RDD002)
  24. KOH
  25. Triton X-100(Sigma-Aldrich,目录号:T9284)
  26. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  27. 无EDTA蛋白酶抑制剂混合物(Roche Diagnostics,目录号:05892953001)
  28. DTT(DL-二硫苏糖醇)(Sigma-Aldrich,目录号:43817)
  29. Tris-HCl(Sigma-Aldrich,目录号:V900312)
  30. NP-40(Sigma Aldrich,目录号:NP40S)
  31. RNase抑制剂(RNaseOUT)(Thermo Fisher Scientific,Invitrogen TM,目录号:10777019)。
  32. 氯化钠(NaCl)(Sigma-Aldrich,目录号:V900312)
  33. 焦碳酸二乙酯(Sigma-Aldrich,目录号:40718)
  34. 本田缓冲区(见食谱)
  35. 高盐核裂解缓冲液(见食谱)
  36. 稀释缓冲液(见食谱)
  37. IP缓冲区(请参阅食谱)


  1. Eppendorf移液器套件(1ml,0.2ml,0.02ml,0.01ml和0.0025ml)
  2. Thermomixer C(Eppendorf,型号:Thermomixer®C,目录号:5382000023)
  3. 真空泵(FJC,型号:6912)
  4. 旋转器(Glas-Col,型号:099A MR1512)
  5. 离心机(Eppendorf,型号:5424 R和5804 R)
  6. Pico Ultrasonicator(Diagenode,型号:Bioruptor Pico,型号:4486126)
  7. 涡旋(Kylin-Bell Lab Instruments,型号:VORTEX-5)


  1. 材料准备
    用4-6周龄的N进行根癌农杆菌介导的相应蛋白质和RNA的瞬时共表达。本生烟。 N。本生烟叶(〜8g)。
    以3 dpi(接种后天数)采集样本。

  2. 核分离
    1. 交叉链接 N。用1%PBS中的1%甲醛处理本生叶15分钟。
    2. 加入甘氨酸至最终浓度为0.125M,充分混合缓冲液并施加真空5分钟以停止交联。
    3. 用无菌水冲洗植物三次。去除多余的水分,将植物冻结在液氮中。如果不立即进入下一步,请将冷冻的植物储存在-80°C。
    4. 用研钵中的液氮将植物材料研磨成细粉。将电源转移到50 ml离心管中。
    5. 对于每个样品(2克),加入15毫升本田缓冲液,在4°C旋转5分钟。
    6. 通过一层Miracloth过滤,然后通过加入另外10ml本田缓冲液(在50ml离心管中)洗涤Miracloth。
    7. 在4℃下以3,500×g 离心5分钟。丢弃上清液,用1毫升本末端缓冲液轻轻悬浮于1毫升本田缓冲液中。
    8. 在3500gxg离心5分钟,用1ml本田缓冲液再次洗涤沉淀。在10000×g下离心1分钟,彻底除去上清液。

  3. 免疫沉淀
    1. 通过使用Diagenode Bioruptor,在核小球中加入2.5体积/重量的高盐核裂解缓冲液,重悬细胞核并超声处理(30秒长脉冲,30秒间隔,10次循环)。
    2. 离心(16,000×g,4℃)10分钟,取上清液(400μl)置于2ml离心管中,加入四倍体积的稀释缓冲液,充分混合,取出1/20,并且保持它作为输入。
    3. 加入抗Myc(10μg/ 2 ml)抗体,4°C旋转2 h,然后加入50μlProtein G并再旋转2 h。
    4. 在4℃下以1,500×g 离心5分钟。小心取出上清液并加入IP缓冲液。用IP缓冲液清洗珠子3次。
    5. 取出1/10的IP提取物进行Western blot检测。
    6. 在4℃下以1,500×g 离心5分钟,彻底除去上清液。

  4. RNA提取和RT-PCR

    1. 加入1 ml Trizol,65°C 10分钟。
    2. 加入200微升氯仿,涡旋,在冰上孵育5分钟。

    3. 以16,000×g 离心5分钟
    4. 取400μl水相。
    5. 加入1微升GlycoBlue和40微升醋酸钠,然后加入1毫升无水乙醇,在-80°C孵育2小时。

    6. 在16,000×g g离心5分钟,弃去上清液。

    7. 在89μl不含核酸酶的水中悬浮RNA沉淀

    8. 加入10μl10x DNase I缓冲液和1μlDNase I,37°C 10分钟
    9. 加300μl无核酸酶的水,然后加入400μl氯仿,涡旋。

    10. 以16,000×g 离心5分钟
    11. 取400μl水相。
    12. 加入1微升GlycoBlue和40微升醋酸钠,然后加入1毫升无水乙醇,-80°C,2小时。

    13. 在16,000×g g离心5分钟,弃去上清液。

    14. 在20μl无核酸酶水中悬浮RNA沉淀
    15. SuperScriptIII反转录产物(按照制造商的说明书)用基因特异性引物进行反转录,并通过RT-PCR(熔解温度:95℃,退火温度:60℃,循环数:30个循环)检测RNA。


使用基因特异性引物逆转录Co-IP产物。通过在2%琼脂糖凝胶上电泳分离逆转录物。如图1所示,OsDRB1a和mOsDRB1a都与apri-miR528相互作用,但均未识别mapri-miR528; NS3和mNS3与OsDRB1a而不是mOsDRB1a相关。随着NS3的表达,OsDRB1a和mOsDRB1a与apri-miR528相互作用。然而,随着mNS3的表达,只有mOsDRB1a与apri-miR528相关。这些结果表明NS3可能作为介导OsDRB1和pri-miRNA之间相互作用的支架。

图1. NS3充当DRB1和pri-miRNA之间的支架RT-PCR检测瞬时共表达蛋白(DRB1a或mDRB1a),pri-miRNA(aprimiR528或mapri)的免疫共沉淀和输入产物-miR528)和蛋白质(空载体,NS3或mNS3)。本塞姆氏。有关更多信息,请参阅Zheng 等。,2017。


  1. 保留所有无RNase的缓冲液。
  2. 如果你有很多样品,一个接一个地分离细胞核,并在液氮中冻结核颗粒。
  3. 关于细胞核分离和免疫沉淀的更详细的信息可以从Saleh等人,2008和Terzi等人,2009年获得。


  1. 本田缓冲区
    0.44 M蔗糖
    20 mM HEPES-KOH,pH7.4
    0.5%Triton X-100
    10mM MgCl 2 2/2 20 U / ml RNA酶抑制剂
    1x鸡尾酒Roche cOmplete(新添加)
  2. 高盐核裂解缓冲液
    20mM Tris-HCl,pH = 7.5
    500 mM NaCl
    4mM MgCl 2 2/2 20U / ml RNA酶抑制剂(新添加)
  3. 稀释缓冲液
    20mM Tris-HCl,pH = 7.5
    4mM MgCl 2 2/2 0.2%NP-40(新添加)
    20U / ml RNA酶抑制剂(新添加)
  4. IP缓冲区
    20mM Tris-HCl,pH = 7.5
    100 mM NaCl
    4mM MgCl 2 2/2 0.2%NP-40(添加新鲜)
    20U / ml RNA酶抑制剂(添加新鲜)


这项工作得到了JG的资助。国家自然科学基金(编号31722045; 31772128; 31701757和31201491)的W.和L. Y.国家基础研究计划973(2014CB138400);和福建省自然科学基金,福建省新世纪优秀青年科学研究计划和优秀人才计划(JA3091,2014J06011)。该协议已于2008年由Saleh et al。和Terzi et al。修改。我们声明不存在利益冲突或利益冲突。


  1. Saleh,A.,Alvarez-Venegas,R.和Avramova,Z。(2008)。 用于研究拟南芥中组蛋白修饰的高效染色质免疫沉淀(ChIP)方案 植物。 Nat Protoc 3(6):1018-1025。
  2. Terzi,L.C.和Simpson,G.G。(2009)。 Arabidopsis RNA免疫沉淀。 Plant J < / em> 59(1):163-168。
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引用:Zheng, L., Zhang, C., Wu, J. and Li, Y. (2018). Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta. Bio-protocol 8(9): e2840. DOI: 10.21769/BioProtoc.2840.