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Isolation of Polysome-bound mRNA from Rice Solid Tissues Amenable for RT-PCR and Profiling Experiments
分离水稻固体组织的多核糖体结合mRNA用于RT-PCR和图谱实验   

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Abstract

Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.

Materials and Reagents

  1. 30-day-old rice roots or shoots
  2. Liquid nitrogen
  3. 3 M sodium acetate (Thermo Fisher Scientific, catalog number: S209-500 )
  4. Chloramphenicol (50 mg/ml) (Sigma-Aldrich, catalog number: C0378 )
  5. Cycloheximide (50 mg/ml) (Sigma-Aldrich, catalog number: C7698 ) in MilliQ water
    Note: Cycloheximide inhibits protein synthesis by blocking translation elongation. This molecule interferes with the translocation of tRNAs with the mRNA and the ribosome, resulting in fixed ribosomes on mRNAs.
  6. 8 M guanidine hydrochloride solution (Sigma-Aldrich, catalog number: G9284 )
  7. Chloroform
  8. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  9. Isopropanol
  10. KCl (Thermo Fisher Scientific, catalog number: BP-366-500 )
  11. Linear acrylamide (5 mg/ml) (Life Technologies, InvitrogenTM, catalog number: AM9520 )
  12. MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
  13. Nonidet P40 (Biocompare, catalog number: DG514 )
  14. Phenol water saturated
  15. Sucrose (Sigma-Aldrich, catalog number: S1888 )
  16. Tris.HCl (pH 8.4) (Thermo Fisher Scientific, catalog number: BP152-1 )
  17. Trizol reagent (Life Technologies, InvitrogenTM, catalog number: 15596-026 )
  18. 10x sucrose solution salts buffer (see Recipes)
  19. 2 M sucrose (see Recipes)
  20. 20-50 % sucrose solutions (see Recipes)
  21. Polysome buffer (see Recipes)

Equipment

  1. 0.22 μm filter
  2. Density gradient fractionation system composed by
    1. Peristaltic pump (Gilson Minipuls 3 peristaltic pump)
    2. Cary 60 UV-Vis Spectrophotometer (Agilent)
    3. Flow cell quartz (1 mm 113 μl, 1/pk) (Agilent, catalog number: 6610019900 )
    4. Fraction collector (Bio-Rad Laboratories, model: 2110 )
  3. Beckman optima L-70 ultracentrifuge (Beckman Coulter)
  4. Beckman tubes
  5. Parafilm
  6. Boekel scientific orbitron rotator I (115V) (Boekel Scientific)
  7. Mini centrifuge (Eppendorf, model: 5415D)
  8. NanoDrop (Thermo Fisher Scientific)

Softawre

  1. The Agilent Cary WinUV software

Procedure

  1. Preparation of sucrose gradients
    1. Prepare 50 ml of 50% (w/v) sucrose solution by mixing 36.7 ml of sucrose (2 M) and 13.3 ml of 10x sucrose salts. Filter solutions with 0.22 μm filter.
    2. Prepare 50 ml of 35% (w/v) sucrose solution by mixing 25.6 ml of sucrose (2 M) and 24.4 ml of 10x sucrose salts. Filter solutions with 0.22 μm filter.
    3. Prepare 62 ml of 20% (w/v) sucrose solution by mixing 18.2 ml of sucrose (2 M) and 43.8 ml of 10x sucrose salts. Filter solutions with 0.22 μm filter.
    4. For one gradient, carefully add 1.85 ml of the 50% sucrose solution, 3.65 ml of the 35%, 3.65 ml of the 20% (w/v) and again 1.35 ml of 20% (w/v) to Beckman tubes (12 ml total volume). Prepare gradients at least one day before cell lysis to allow gradient to diffuse overnight at 4 °C.
    5. Cover the tube with parafilm and incubate at 4 °C overnight. Alternatively, sucrose gradients can be stored at -80 °C indefinitely.


      Figure 1. Sucrose gradients. Distribution of the sucrose layers (sucrose concentrations shown as percent weight per volume on right).

  2. Isolation and polysome profiling
    1. 30-day-old rice roots or shoots were harvested, frozen and ground to powder in liquid nitrogen.
    2. 150 mg of powder was combined with 1.2 ml of chilled polysome buffer.
    3. Debris was removed by centrifugation at 16,000 x g for 15 min at 4 °C.
    4. Aliquots of the resulting supernatant were transferred (about 500 μl) to a new pre-chilled 1.5 ml tube. Measure OD260nm for each sample using NanoDrop.
    5. Load the same OD amount of lysate onto each gradient on to 20 to 50% (w/w) continuous sucrose gradients. Keep 10% of lysates as an input to determine the cytosolic steady-state mRNA levels.
    6. Weight and balance each gradient before ultracentrifugation.
    7. Centrifuge at 175,000 x g for 165 min at 4 °C using SW41Ti rotor in a Beckman Coulter.
    8. While the samples are centrifuging, clean fraction collector with warm MilliQ water containing a bit of RNase decontamination solution.
    9. Carefully remove tubes from the rotor and place them at 4 °C until they are ready for running.
    10. Switch on computer, pump, UV-Vis Spectrophotometer and fraction collector. Set pump at 2.3 ml/min and the fraction collector by time. Place 10 ml tubes on fraction collector.
    11. Open Cary WinUV software.
    12. Fractions were collected from the bottom to the top of the gradient with continuous monitoring of the absorbance at 254 nm. Run chasing solution [20% (w/v) sucrose] with bromophenol blue through the system until it reaches the needle. Make sure to see at least one drop coming out of the needle such that no bubbles are introduced into the gradient.
    13. Begin running the chasing solution through the gradient. Run solution with the pump at 2.3 ml/min.
    14. Click on acquire data button and press run on the fraction collector.
    15. Place fractions on dry ice.

  3. Polysomal RNA extraction
    1. Add 4 ml of Trizol reagent to each tube
    2. Isolate RNA according to manufacturer’s Trizol protocol.
    3. Measure the RNA concentration of each fraction or input.

Representative data

We used a simple and highly reproducible method for the analysis of polysomes in rice (Oryza sativa). All the experiments we made, displayed a very similar profiles, however, some differences can be observed in the form of the profiles when the plants are stressed and also in the older plants. In the literature, analysis of polysome profiles issued from different plant species show a variability between species and plant organs as shown in Figure 2 below.




Figure 2. Analysis of polysome profiles from different plant species

Example 1 (Figure 2a): Polysomal extrcation from Medicago truncatula roots. Ribosomes were pelleted by ultracentrifugation of whole extracts (S-16) of transgenic roots through a 1.7-m sucrose cushion to obtain the post-ribosomal supernatant (S-170) and the ribosome pellet (P-170). P-170 was fractionated by ultracentrifugation through a 20–60% (w/v) sucrose density gradient and the absorbance at 254 nm was recorded. Positions of monosomes (80S) and large polysomes (LP, ≥5 ribosomes) are indicated. [from Reynoso et al. (2012)]
Example 2 (Figure 2b-c): Ribosomes purified from Arabidopsis tahliana leaf tissues by conventional ultracentrifugation (P-170) (a) or by immunopurification (eluate) (b) were fractionated in 20% to 60% (w/v) sucrose density gradients and the UV absorbance (254 nm) profile was recorded. The positions of the 60S ribosomal subunit, 80S mono-somes, and polysomes are indicated. [from Zanetti et al. (2005)]
Example 3 (Figure 2d): Cadmium-induced alteration of the polysomes profile using Arabidopsis thaliana suspension cells . The polysome profile of control and cadmium-treated cells treated with 200 μM cadmium for 4 or 12 h, are represented. [from Sormani et al. (2011)]
Example 4 (Figure 2e): Sucrose density profiles of ribosomes and polysomes from Pisum sativum. Ribosomes recovered after 6 h. centrifugation through 1 M-sucrose cushion. E254=absorbance at 254 nm.
[from Leaver and Dyer (1974)]
Example 5 (Figure 2f-g): Typical polysome profiles obtained from tobacco leaf tissues and protoplasts, respectively, are shown in (a). The effect of detachment on the polysome populations of tobacco leaves is schown un (b). Sucrose density gradient profiles of polysome preparations obtained from attached leaves (left), leaves detached and floated on water for 4 h (middle) and leaves detached and floated on 0.6 M mannitol for 4 h (right). [from Ruzicska et al. (1979)]
Example 6 (Figure 2h): Polysome profiles generated from roots of young WT rice plants grown in Pi-sufficient media (15 d-old). [from Jabnoune (unpublished)]


Figure 3. Fractionation of polysome gradients


Figure 4. A representative example of data obtained

Note: We used a simple and highly reproducible method for the analysis of polysomes in rice (Oryza sativa). All the experiments we made, displayed a very similar profiles, however, some differences can be observed in the form of the profiles when the plants are stressed and also in the older plants. In the literature, analysis of polysome profiles issued from different plant species show a variability between species and plant organs (see examples in Figure 2).

Notes

To isolate the RNA form fractions from sucrose density centrifugation for polysome analysis, it’s important to keep in mind that TRizol purification could be the best option in case of large scale extraction (Rice produce more biological materiel than Arabidospis). In case of small samples (particularly Arabidopsis roots samples) the use of RNeasy kit purification of RNA will be more cost-effective but sometimes the quality of the RNA would be not of high quality for subsequent qPCR analysis. It’s important to centrifuge the Trizol extract at 12,000 x g for 15 min to separate the phases. We have to sacrifice a layer of the aqueous phase (containing RNA) at the interphase, so that we will carry over proteins and phenol to the RNA.

Recipes

  1. 10x sucrose solution salts buffer (100 ml)
    0.4 M Tris-HCl (pH 8.4)
    0.2 M KCl
    0.1 M MgCl2
    qsq 100 ml H2O
    Autoclave
    Add after autoclaving
    100 μg/ml cycloheximide
    1x protease inhibitor cocktail (EDTA-free)
    100 units/ml RNase inhibitor
  2. 2 M sucrose (200 ml)
    Sucrose 137 g
    Qsq 200 ml 1x sucrose solution salts
    Autoclave
  3. 20-50 % sucrose solutions

    Final (%)
    Volume (ml)
    Sucrose 2 M
    1x sucrose salts
    50 %
    12
    8.8
    3.2

    25
    18.3
    6.7

    50
    36.7
    13.3

    75
    55
    20
    35%
    25
    12.9
    12.1

    45
    23.1
    21.9

    50
    25.6
    24.4

    90
    46.3
    43.7

    100
    51.3
    48.7

    150
    76.9
    73.1
    20%
    32
    9.4
    22.6

    62
    18.2
    43.8

    122
    35.8
    86.2

    200
    58.6
    141.4

  4. Polysome buffer (10 ml)
    10x sucrose solution salts 2.5 ml
    EGTA 20 mg
    Nonidet-P40 50 µl
    Cycloheximide (50 mg/ml) 10 µl
    Chlormaphenicol (50 mg/ml) 10 µl
    Qsq 10 ml H2O

Acknowledgments

This work was funded by the Swiss National Foundation grant (31003A-12293 and 31003A-138339) and the Sino-Swiss Science and Technology Cooperation Program (IZLCZ3 123946 to YP and 2009DFA32040 to QS).

References

  1. Dowling, R. J., Topisirovic, I., Alain, T., Bidinosti, M., Fonseca, B. D., Petroulakis, E., Wang, X., Larsson, O., Selvaraj, A., Liu, Y., Kozma, S. C., Thomas, G. and Sonenberg, N. (2010). mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328(5982): 1172-1176.
  2. Lanet, E., Delannoy, E., Sormani, R., Floris, M., Brodersen, P., Crete, P., Voinnet, O. and Robaglia, C. (2009). Biochemical evidence for translational repression by Arabidopsis microRNAs. Plant Cell 21(6): 1762-1768.
  3. Leaver, C. J. and Dyer, J. A. (1974). Caution in the interpretation of plant ribosome studies. Biochem J 144(1): 165-167.
  4. Reynoso, M. A., Blanco, F. A., Bailey-Serres, J., Crespi, M. and Zanetti, M. E. (2012). Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula. Plant J 73: 289-301.
  5. Ruzicska, P., Mettrie, R., Dorokhov, Y. L., Premecz, G., Olah, T. and Farkas, G. L. (1979). Polyribosomes in protoplasts isolated from tobacco leaves. Planta 145(2): 199-203.
  6. Sormani, R., Delannoy, E., Lageix, S., Bitton, F., Lanet, E., Saez-Vasquez, J., Deragon, J. M., Renou, J. P. and Robaglia, C. (2011). Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels. Plant Cell Physiol 52(2): 436-447.
  7. Zanetti, M. E., Chang, I. F., Gong, F., Galbraith, D. W. and Bailey-Serres, J. (2005). Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiol 138(2): 624-635.
    16532-K

简介

Polysome谱分析是翻译控制研究中经常执行的任务,不仅能够直接监测翻译的效率,而且可以很容易地扩展到广泛的下游应用,如Northern和western印迹,全基因组微阵列分析或qRT- PCR。 在这里,我们描述了一种从少量液氮冷冻固体组织样本的水稻芽/根中分离和定量高质量多糖结合mRNA复合物的方法。 获得的mRNA可以通过在单个转录物或全局水平评估多核糖体mRNA丰度的方法进一步分析。

材料和试剂

  1. 30日龄的水稻根或芽
  2. 液氮
  3. 3 M乙酸钠(Thermo Fisher Scientific,目录号:S209-500)
  4. 氯霉素(50mg/ml)(Sigma-Aldrich,目录号:C0378)
  5. 在MilliQ水中的环己酰亚胺(50mg/ml)(Sigma-Aldrich,目录号:C7698)
    注意:环己酰亚胺通过阻断翻译延伸来抑制蛋白质合成。 该分子干扰tRNA与mRNA和核糖体的移位,导致mRNA上的固定核糖体。
  6. 8M盐酸胍溶液(Sigma-Aldrich,目录号:G9284)
  7. 氯仿
  8. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N' - 四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  9. 异丙醇
  10. KCl(Thermo Fisher Scientific,目录号:BP-366-500)
  11. 线性丙烯酰胺(5mg/ml)(Life Technologies,Invitrogen TM,目录号:AM9520)
  12. MgCl 2(Sigma-Aldrich,目录号:M-2393)
  13. Nonidet P40(Biocompare,目录号:DG514)
  14. 苯酚水饱和
  15. 蔗糖(Sigma-Aldrich,目录号:S1888)
  16. Tris缓冲液(pH8.4)(Thermo Fisher Scientific,目录号:BP152-1)
  17. Trizol试剂(Life Technologies,Invitrogen TM,目录号:15596-026)
  18. 10x蔗糖溶液盐缓冲液(见配方)
  19. 2 M蔗糖(见配方)
  20. 20-50%蔗糖溶液(见配方)
  21. 聚合物缓冲液(参见配方)

设备

  1. 0.22μm过滤器

  2. 组成的密度梯度分馏系统
    1. 蠕动泵(Gilson Minipuls 3蠕动泵)
    2. Cary 60紫外 - 可见分光光度计(Agilent)
    3. 流动池石英(1mm113μl,1/pk)(Agilent,目录号:6610019900)
    4. 馏分收集器(Bio-Rad Laboratories,型号:2110)
  3. Beckman optima L-70超速离心机(Beckman Coulter)
  4. Beckman管
  5. parafilm
  6. Boekel科学轨道旋转器I(115V)(Boekel Scientific)
  7. Mini离心机(Eppendorf,型号:5415D)
  8. NanoDrop(Thermo Fisher Scientific)

软件

  1. Agilent Cary WinUV软件

程序

  1. 蔗糖梯度的制备
    1. 通过混合36.7ml蔗糖制备50ml 50%(w/v)蔗糖溶液   (2M)和13.3ml 10x蔗糖盐。 0.22μm过滤器 过滤器
    2. 通过混合制备50ml的35%(w/v)蔗糖溶液 25.6ml蔗糖(2M)和24.4ml 10x蔗糖盐。 过滤 具有0.22μm过滤器的溶液
    3. 制备62ml的20%(w/v)蔗糖   溶液,通过混合18.2ml蔗糖(2M)和43.8ml 10x蔗糖 盐。 用0.22μm过滤器过滤溶液。
    4. 对于一个梯度, 小心地加入1.85ml的50%蔗糖溶液,3.65ml的35% 3.65ml的20%(w/v)和再次1.35ml的20%(w/v)的Beckman管   (12ml总体积)。 至少在细胞前一天准备梯度 裂解使梯度在4℃下扩散过夜
    5. 封面 用石蜡膜封闭,并在4℃下孵育过夜。 或者, 蔗糖梯度可以无限期储存在-80℃

      图1。 蔗糖梯度。蔗糖层的分布(蔗糖 浓度显示为右侧的每体积重量百分比)。

  2. 隔离和多核糖体分析
    1. 收获30日龄的稻根或芽,冷冻并在液氮中研磨成粉末
    2. 将150mg粉末与1.2ml冷冻多核糖体缓冲液混合。
    3. 通过在4℃下以16,000×g离心15分钟除去碎片。
    4. 转移所得上清液的等分试样(约500μl) μl)至新的预冷的1.5ml管中。 对于每个样品测量OD 260nm 使用NanoDrop。
    5. 加载相同OD量的裂解物到每个 梯度至20至50%(w/w)连续蔗糖梯度。 保持10%   裂解物作为输入以确定胞质稳态mRNA 级别。
    6. 在超速离心之前,每个梯度重量和平衡
    7. 在4℃下使用SW41Ti转子在Beckman Coulter中以175,000×g离心165分钟。
    8. 当样品离心时,用部分收集器清洗 温的MilliQ水,含有一点RNase净化溶液
    9. 小心地从转子上取下管子,并将其放置在4°C,直到它们准备好运行
    10. 打开计算机,泵,UV-Vis分光光度计和分数 集电极。 按2.3 ml/min设置泵,按时间设置馏分收集器。 将10ml管置于馏分收集器上。
    11. 打开Cary WinUV软件。
    12. 从梯度的底部到顶部收集级分   连续监测254nm处的吸光度。 运行追逐 溶液[20%(w/v)蔗糖]与溴酚蓝反应 直到它到达针。 确保看到至少一滴下降 离开针头,使得没有气泡被引入到梯度中
    13. 开始通过梯度运行追逐解决方案。 使用泵以2.3ml/min运行溶液
    14. 点击获取数据按钮,并在馏分收集器上运行
    15. 将馏分置于干冰上。

  3. 多聚体RNA提取
    1. 向每个管中加入4 ml Trizol试剂
    2. 根据制造商的Trizol方案分离RNA
    3. 测量每个馏分或输入的RNA浓度。

代表数据

我们使用一种简单和高度可重复的方法来分析水稻中的多核糖体( Oryza sativa )。 我们做的所有实验,显示非常相似的配置文件,但是,当植物压力和老植物时,可以观察到轮廓的形式的一些差异。 在文献中,来自不同植物物种的多核糖体谱的分析显示物种和植物器官之间的变异性,如下面的图2所示。




图2.来自不同植物物种的多核糖体谱的分析

实施例1(图2a):来自Medic藜苜蓿根的多粒体诱发。通过1.7-m蔗糖垫超速离心转基因根的全部提取物(S-16)以获得核糖体上清液(S-170)和核糖体沉淀物(P-170),使核糖体沉淀。通过20-60%(w/v)蔗糖密度梯度超速离心分离P-170,记录254nm处的吸光度。显示了单体(<80mS)和大多核糖体(LP,≥5个核糖体)的位置。 [来自Reynoso 等人。 (2012)]
实施例2(图2b-c):通过常规超速离心(P-170)(a)或通过免疫纯化(洗脱液)(b)从天冬瓜拟南芥叶组织中纯化的核糖体在20%至60记录%(w/v)蔗糖密度梯度和UV吸光度(254nm)分布。显示了60S核糖体亚基,80S单体和多核糖体的位置。 [来自Zanetti 等人。 (2005)]
实施例3(图2d):镉诱导的使用拟南芥悬浮细胞的多核糖体谱的改变。用200μM镉处理4或12小时的对照和镉处理的细胞的多核糖体谱。 [from Sormani et al 。 (2011)]
实施例4(图2e):来自豌豆的核糖体和多核糖体的蔗糖密度图。 6小时后回收核糖体。通过1M蔗糖垫离心。 E254 =在254nm处的吸光度 [来自Leaver和Dyer(1974)]
实施例5(图2f-g):分别从烟草叶组织和原生质体获得的典型多核糖体谱显示在(a)中。分离对烟草叶的多核糖体群的影响显示为un(b)。从附着叶(左)获得的多核糖体制剂的蔗糖密度梯度曲线,叶分离并漂浮在水上4小时(中间),叶子分离并漂浮在0.6M甘露醇上4小时(右)。 [来自Ruzicska ]。 (1979)]
实施例6(图2h):从在充足的培养基(15天龄)中生长的幼小WT水稻植物的根产生的聚合体轮廓。 [来自Jabnoune(未发表)]


图3.多核糖梯度的分馏


图4.获取的数据的代表示例

注意:我们使用一种简单和高度可重复的方法来分析水稻(Oryza sativa)中的多核糖体。我们做的所有实验,显示非常相似的配置文件,但是,当植物压力和老植物时,可以观察到轮廓的形式的一些差异。在文献中,来自不同植物物种的多核糖体谱的分析显示物种和植物器官之间的变异性(参见图2中的实例)。

笔记

为了从蔗糖密度离心中分离RNA形式级分用于多核糖体分析,重要的是要记住,在大规模提取的情况下(稻产生比Arabidospis更多的生物材料),TRizol纯化可以是最佳选择。在小样品(特别是拟南芥根样品)的情况下,使用RNeasy试剂盒纯化RNA将更具成本效益,但有时RNA的质量对于随后的qPCR分析将不是高质量的。重要的是,以12,000×g离心Trizol提取物15分钟以分离各相。我们必须在中间相处牺牲一层水相(含有RNA),以便我们将蛋白质和苯酚带到RNA。

食谱

  1. 10x蔗糖溶液盐缓冲液(100ml) 0.4M Tris-HCl(pH 8.4)
    0.2 M KCl
    0.1M MgCl 2
    qsq 100 ml H 2 O v/v 高压灭菌器
    高压灭菌后加入
    100μg/ml放线菌酮
    1x蛋白酶抑制剂混合物(无EDTA)
    100单位/ml RNase抑制剂
  2. 2M蔗糖(200ml) 蔗糖137g
    Qsq 200ml 1x蔗糖溶液盐
    高压灭菌器
  3. 20-50%蔗糖溶液
    最终(%)
    体积(ml)
    蔗糖2μM/ 1x蔗糖盐
    50%
    12
    8.8
    3.2

    25
    18.3
    6.7

    50
    36.7
    13.3

    75
    55
    20
    35%
    25
    12.9
    12.1

    45
    23.1
    21.9

    50
    25.6
    24.4

    90
    46.3
    43.7

    100
    51.3
    48.7

    150
    76.9
    73.1
    20%
    32
    9.4
    22.6

    62
    18.2
    43.8

    122
    35.8 / 86.2

    200
    58.6
    141.4

  4. 聚合体缓冲液(10ml)
    10x蔗糖溶液盐2.5ml
    EGTA 20mg
    Nonidet-P40 50微升
    环己酰亚胺(50mg/ml)10μl
    氯霉素(50mg/ml)10μl
    Qsq 10ml H 2 O *

致谢

这项工作由瑞士国家基金会拨款(31003A-12293和31003A-138339)和中瑞科学技术合作计划(IZLCZ3 123946到YP和2009DFA32040到QS)资助。

参考文献

  1. Dowling,RJ,Topisirovic,I.,Alain,T.,Bidinosti,M.,Fonseca,BD,Petroulakis,E.,Wang,X.,Larsson,O。,Selvaraj,A.,Liu,Y.,Kozma, SC,Thomas,G。和Sonenberg,N。(2010)。 mTORC1介导的细胞增殖,但不是细胞生长,由4E-BP控制。 Science 328(5982):1172-1176。
  2. Lanet,E.,Delannoy,E.,Sormani,R.,Floris,M.,Brodersen,P.,Crete,P.,Voinnet,O.and Robaglia, 拟南芥微RNA的翻译抑制的生物化学证据。 em> Plant Cell 21(6):1762-1768
  3. Leaver,C.J。和Dyer,J.A。(1974)。 注意植物核糖体研究的解释 Biochem J 144(1):165-167。
  4. Reynoso,M.A.,Blanco,F.A.,Bailey-Serres,J.,Crespi,M。和Zanetti,M.E。 选择性地将mRNA和miRNA选择性地募集到多核糖体中以响应根瘤菌感染在Medic藜苜蓿 73:289-301。
  5. Ruzicska,P.,Mettrie,R.,Dorokhov,Y.L.,Premecz,G.,Olah,T.and Farkas,G.L。(1979)。 从烟草叶中分离的原生质体中的聚核糖体。 145 (2):199-203
  6. Sormani,R.,Delannoy,E.,Lageix,S.,Bitton,F.,Lanet,E.,Saez-Vasquez,J.,Deragon,J.M.,Renou,J.P.and Robaglia, 拟南芥亚致死性镉中毒在多个层面影响翻译。植物细胞Physiol 52(2):436-447
  7. Zanetti,M.E.,Chang,I.F.,Gong,F.,Galbraith,D.W。和Bailey-Serres,J。(2005)。 拟南芥的多核糖体复合物的免疫纯化用于基因表达的全局分析植物 Physiol 138(2):624-635。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q. and Poirier, Y. (2015). Isolation of Polysome-bound mRNA from Rice Solid Tissues Amenable for RT-PCR and Profiling Experiments. Bio-protocol 5(5): e1411. DOI: 10.21769/BioProtoc.1411.
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