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5’ Rapid Amplification of cDNA Ends (5’ RACE) of Agrobacterial T-DNA Genes within Transformed Plant Sample
对转基因植株样本中的农杆菌上T-DNA基因的cDNA 5'末端进行快速扩增(5'RACE)   

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Abstract

The T-DNA (transferred-DNA) region of virulent Agrobacterium tumefaciens (A. tumefaciens) strain is transferred and integrated into the plant genome, and thereby the T-DNA genes are expressed in transformed plant cells. This protocol was used to analyze the transcription start sites (TSSs) of agrobacterial T-DNA genes within plant crown gall tumor. Firstly, the stems of Arabidopsis thaliana were inoculated by A. tumefaciens strain C58 and developed crown gall tumor. Subsequently, the mRNA was extracted from the crown gall tumor and then used for amplification of 5’ cDNA ends by 5’ Rapid Amplification of cDNA Ends (5’ RACE) assay. The full-length cDNAs were generated in reverse transcription reactions and used to analyze TSSs. Here, TSSs of three oncogenes, IaaH, IaaM and Ipt were analyzed as examples. This protocol also allows for identification of TSSs of the other agrobacterial T-DNA genes that expressed in plant cells.

Keywords: Agrobacterium tumefaciens(农杆菌介导法), T-DNA(T-DNA), 5' RACE(5的比赛), Transcription start site(转录起始位点)

Materials and Reagents

  1. A. tumefaciens strain C58 nocc (nopaline catabolism) (MAX-PLANCK-GESELLSCHAFT, catalog number: 584 )
  2. Arabidopsis thaliana Col-0
  3. Escherichia coli (E. coli) strain MRF’ (Agilent Technologies, catalog number: 200230-41 )
  4. Dynabeads® Oligo(dT)25 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 61005 )
  5. SMARTer® RACE 5’/3’ Kit (Takara Bio Company, Clontech, catalog number: 634859 )
  6. DreamTaq DNA Polymerase (5 U/µl) (Thermo Fisher Scientific, catalog number: EP0701 )
  7. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
  8. pGEM®-T Easy Vector Systems (Promega Corporation, catalog number: A1360 )
  9. Hypodermic Syringes without Needle (5 cc) (Terumo Medical Corporation, catalog number: SS-05L )
  10. Hypodermic Needles (Terumo Medical Corporation, catalog number: NN-2138R )
  11. KB medium (see Recipes)
  12. Agromix buffer (see Recipes)
  13. Lysis buffer (see Recipes)
  14. Washing buffer A (see Recipes)
  15. Washing buffer B (see Recipes)
  16. Elution buffer (see Recipes)
  17. 2x binding buffer (see Recipes)

Equipment

  1. Culture tubes 13 ml (SARSTEDT, catalog number: 62.515.028 )
  2. NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: ND-2000c )
  3. High-performance, Modular Stereomicroscope for Application Based Customization MZ6 (Leica Microsystems, model: MZ6)
  4. Ball mill (RETSCH, model: MM200 )
  5. Thermal cycler (Eppendorf, catalog number: 950000031 )
  6. MiniSpin®/MiniSpin® plus Centrifuge (Eppendorf, catalog number: 0 22620100 )
  7. MagneSphere® Technology Magnetic Separation Stands (Promega Corporation, catalog number: Z5342 )

Procedure

  1. Crown gall tumor induction
    1. Overnight culture of A. tumefaciens was obtained from colonies grown on KB agar plates. Colonies were transferred into 3-5 ml KB liquid medium in 13 ml culture tubes with assembled two-position ventilation cap (with position 1: Cap lightly pushed on, ventilated) and incubated at 140 rpm in a rotary shaker at 28 °C for overnight.
    2. Overnight A. tumefaciens cells were centrifuged at 8,000 rpm for 1 min and suspended in 3 ml agromix buffer. Suspended cells were cultured at 28 °C, 140 rpm again for 2-3 h. OD600 was measured by spectrophotometer and adjusted to OD600 = 0.5.
    3. Young inflorescence stems (3 to 10 cm) of Arabidopsis were injected near the bottom with a 5 ml syringe two times at one direction and two times at another perpendicular direction (Figure 1). The syringe needle was injected completely through the stems. One drop of A. tumefaciens cells was used for one injection.


    Figure 1. Schematic image of Arabidopsis influrecence stem. Young inflorescence stems were injected with a syringe in the injection zone (marked). The original Arabidopsis image is from website (http://www.lookfordiagnosis.com/mesh_info.php?term=Arabidopsis&lang=1).

    1. The stems were injected eight times in total, so usually eight blocks of tumors were generated at 25 days after inoculation (Figure 2). The tumors were cut from the stems with a scalpel and tweezer under a dissecting microscope Leica MZ6 and weighed in the balance.


    Figure 2. Arabidopsis crown gall tumors. The crown gall tumors were generated from the Arabidpsis stems at 25 days after inoculation. The tumors (marked by red circle) were cut with a scalpel and tweezer and weighed.

  2. Poly-A-mRNA extraction
    Approximately 50 mg crown gall tumor was used for mRNA extraction. Poly-A-mRNA was isolated by the oligonucleotide Poly-dTTP (25) bound to polystyrene beads. Dynabeads Oligo-[dT] 25 kit was used.
    1. The plant tissue was frozen in liquid nitrogen and homogenized in a ball mill, and then 1 ml Lysis Buffer was added and the samples were further homogenized for 1-2 min at room temperature until the tissue was completely lysed. The lysate was centrifuged for 30 sec at 10,000 rpm.
    2. The manufacturer’s protocol of Dynabeads Oligo-[dT] 25 kit was performed. The supernatant was incubated for 30 min with 20 µl Dynabeads at room temperature in an overhead rotor. The tubes were place on the MagneSphere® Technology Magnetic Separation Stands for 2 min and removed the supernatant. The manufacturer’s protocol for the magnetic stand was performed.
    3. The first washing step: The Dynabeads were washed twice with 200 µl Washing Buffer A, and twice with 200 µl Washing Buffer B. The magnet was used to separate beads and supernatant for each washing. The Dynabeads were washed by gently flicking the bottom of the tube until all beads were resuspended, and then the beads were captured by magnetic stand. The supernatant was removed without disturbing the beads.
    4. In order to exclude DNA contaminations, the mRNA was eluted from the beads by 50 µl Elution Buffer and incubation at 75 °C for 2 min.
    5. The second washing step: 50 µl 2x Binding Buffer was added to the eluted mRNA, and the 100 µl of mixture was again incubated 20 min at room temperature in an overhead shaker.
    6.  After two washing steps with 200 µl Washing Buffer B, the mRNA was eluted from the magnetic beads with 22 µl Elution Buffer at 75 °C for 2 min. The concentration of mRNA was quantified by NanoDrop 2000c UV-Vis Spectrophotometer. The mRNA was directly used for first-strand cDNA synthesis for 5’ RACE assay or stored at -80 °C.
  3. 5’- rapid amplification of cDNA ends (5’ RACE) assay
    5’ ends of the oncogenes cDNA were amplified by SMARTer™ RACE cDNA Amplification Kit. First-Strand cDNA was generated by SMARTScribe™ Reverse Transcriptase and primed using SMARTer II A Oligonucleotide and 5'-CDS Primer A.
    1. The following reagents were added in one tube
      2.75 µl        mRNA
      1.0 µl 5'-CDS Primer A
      The reagents were mixed well by pipet and incubated the tubes at 72 °C for 3 min, and then 42 °C for 2 min.
    2. The following reagents were to the mixture from step a for a total volume of 10 µl
      1.0 µl         SMARTer II A Oligonucleotide
      2.0 µl         5x First-Strand Buffer
      1.0 µl         DTT (20 mM)
      1.0 µl         dNTP Mix (10 mM)
      0.25 µl       RNase Inhibitor (40 U/μl)
      1.0 µl         SMARTScribe™ Reverse Transcriptase (100 U)
      The reagents were mixed well by pipet and incubated the tubes at 42 °C for 90 min.
    3. The reaction mixture was heated at 70 °C for 10 min to terminate the reaction and the first-strand reaction product was diluted with 10 folds ddH2O. Samples were stored at -20 °C or used for next step directly.
  4. Amplification and sequencing of 5’ ends of the oncogenes cDNA
    1. The fragments of 5’ ends of the oncogenes cDNA were amplified by DreamTaq DNA Polymerase and primed using Universal Primer A Mix (UPM) and specific target primer (Table 1) as shown in the following reaction mixture.
      PCR reaction (a total volume of 50 µl):
      cDNA from step 3.3.1                               1 µl
      5x Buffer                                                10 µl
      10x Universal Primer A Mix                       5 µl
      Specific target reverse primer (10 µM)        1 µl
      dNTP (10 mM)                                         1 µl
      DreamTaq DNA Polymerase                   0.5 µl
      ddH2O to a final volume of                       50 µl
      PCR program:
      Step 1: 95 °C                     1 min
      Step 2: 95 °C                    30 sec
      Step 3: 55 °C                    30 sec
      Step 4: 72 °C                    15 sec
      Step 5: repeat step 2-4 for 30 cycles
      Step 6: 72 °C                     5 min
      Step 7: 4 °C                       Store

    Table 1. Specific oncogene reverse primer
    Gene name (Accession No.)
    Primer sequence (5’ → 3’)
    IaaH (pTiC58, AE007871)
    CCCCGATTGCTAACAGACG
    IaaM (pTiC58, AE007871)
    CAAGAGTGTTCGAGAGG
    Ipt (pTiC58, AE007871)
    TCCCATGAATCAACTTAT

    1. The PCR products were purified by QIAquick PCR Purification Kit.
    2. The purified PCR products were inserted into pGEM-T Easy Vector by TA cloning.
      2x Rapid Ligation Buffer                         5 µl
      pGEM-T Easy Vector (50 ng/ µl)          0.5 µl
      PCR product                                      3.5 µl
      T4 DNA Ligase                                     1 µl
      The reagents were mixed by pipet and incubated for 1 h at room temperature.
    3. The resulting recombinant vectors from step 4c were transformed into E. coli strain MRF’ using the heat shock method.
    4. More than three independent colonies were sequenced for 5’ end of the PCR products, which were the transcription start sites of oncogene.

Representative data

Table 2. Cis-regulatory sequence elements within the oncogene promoters

This data was published on PLOS Pathogens (Zhang et al., 2015). Positive numbers indicate the positions downstream and negative numbers the positions upstream of the TSSs (+1). TSS is underlined. Y = C/T, W = A/T, N = A/G/C/T. 1. (Klee et al., 1984); 2. (Nester et al., 1984); 3. (Goldberg et al., 1984); 4. (Heidekamp et al., 1983); 5. (de Pater et al., 1987); 6. (Lichtenstein et al., 1984)

Recipes

  1. KB medium
    20 g protease peptone
    1.5 g K2HPO4
    0.87% glycerol
    600 µM MgSO4
    15 g agar (plate only)
    Sterilized by autoclave
  2. Agromix buffer
    0.01 M MgCl2
    0.01 M MES
    pH 5.6
  3. Lysis buffer
    100 mM Tris-HCl (pH 7.5)
    500 mM LiCl
    10 mM EDTA
    1% LiDS
    5 mM dithiothreitol (DTT)
    If any precipitation is observed, warm the buffer to room temperature and shake until all the components are fully resuspended.
  4. Washing buffer A
    10 mM Tris-HCl (pH 7.5)
    0.15 M LiCl
    1 mM EDTA
    0.1% LiDS
  5. Washing buffer B
    10 mM Tris-HCl (pH 7.5)
    0.15 M LiCl
    1 mM EDTA
  6. Elution buffer
    10 mM Tris-HCl (pH 7.5)
  7. 2x binding buffer
    20 mM Tris-HCl (pH 7.5)
    1.0 M LiCl
    2 mM EDTA

Acknowledgments

I would like to thank Rosalia Deeken (Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, Germany) for supervision of this work and China Scholarship Council (CSC) for my financial support. I would also like to give many thanks to Shiqiang Gao (Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, Germany) for help, suggestion and sharing reagents.

References

  1. de Pater, B. S., Klinkhamer, M. P., Amesz, P. A., de Kam, R. J., Memelink, J., Hoge, J. H. and Schilperoort, R. A. (1987). Plant expression signals of the Agrobacterium T-cyt gene. Nucleic Acids Res 15(20): 8267-8281.
  2. Goldberg, S. B., Flick, J. S. and Rogers, S. G. (1984). Nucleotide sequence of the tmr locus of Agrobacterium tumefaciens pTi T37 T-DNA. Nucleic Acids Res 12(11): 4665-4677.
  3. Heidekamp, F., Dirkse, W. G., Hille, J. and van Ormondt, H. (1983). Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encode d tmr gene. Nucleic Acids Res 11(18): 6211-6223.
  4. Klee, H., Montoya, A., Horodyski, F., Lichtenstein, C., Garfinkel, D., Fuller, S., Flores, C., Peschon, J., Nester, E. and Gordon, M. (1984). Nucleotide sequence of the tms genes of the pTiA6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis. Proc Natl Acad Sci U S A 81(6): 1728-1732.
  5. Lichtenstein, C., Klee, H., Montoya, A., Garfinkel, D., Fuller, S., Flores, C., Nester, E. and Gordon, M. (1984). Nucleotide sequence and transcript mapping of the tmr gene of the pTiA6NC octopine Ti-plasmid: a bacterial gene involved in plant tumorigenesis. J Mol Appl Genet 2(4): 354-362.
  6. Nester, E. W., Gordon, M. P., Amasino, R. M. and Yanofsky, M. F. (1984). Crown gall - a molecular and physiological analysis. Annual Review Of Plant Physiology And Plant Molecular Biology 35: 387-413.
  7. Zhang, Y., Lee, C. W., Wehner, N., Imdahl, F., Svetlana, V., Weiste, C., Droge-Laser, W. and Deeken, R. (2015). Regulation of oncogene expression in T-DNA-transformed host plant cells. PLoS Pathog 11(1): e1004620.

简介

将有毒的根癌土壤杆菌(根瘤土壤杆菌)菌株的T-DNA(转移的DNA)区转移并整合到植物基因组中,从而T-DNA基因在转化的植物细胞中表达。该方案用于分析植物冠gall肿瘤内农杆菌T-DNA基因的转录起始位点(TSS)。首先,通过接种拟南芥的茎。 tumefaciens 菌株C58和发展的冠gall肿瘤。随后,从冠gall瘤提取mRNA,然后通过cDNA末端的快速扩增(5'RACE)测定用于扩增5'cDNA末端。全长cDNA在逆转录反应中产生并用于分析TSS。这里,作为实例分析了三种癌基因的TSS,IaaH ,IaaM 和 Ipt 。该协议还允许鉴定在植物细胞中表达的其他农杆菌T-DNA基因的TSS

关键字:农杆菌介导法, T-DNA, 5的比赛, 转录起始位点

材料和试剂

  1. A。 (MAX-PLANCK-GESELLSCHAFT,目录号:584),其中所述的菌株C58 nocc(胭脂碱分解代谢)
  2. Arabidopsis thaliana Col-0
  3. 大肠杆菌(大肠杆菌)菌株MRF'(Agilent Technologies,目录号:200230-41)
  4. Dynabeads Oligo(dT)25(Thermo Fisher Scientific,Invitrogen TM,目录号:61005)
  5. SMARTer RACE 5'/3'Kit(Takara Bio Company,Clontech,目录号:634859)
  6. DreamTaq DNA聚合酶(5U /μl)(Thermo Fisher Scientific,目录号:EP0701)
  7. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  8. pGEM -T Easy Vector Systems(Promega Corporation,目录号:A1360)
  9. 无针皮下注射器(5cc)(Terumo Medical Corporation,目录号:SS-05L)
  10. 皮下针(Terumo Medical Corporation,目录号:NN-2138R)
  11. KB介质(参见配方)
  12. Agromix缓冲区(参见配方)
  13. 裂解缓冲液(见配方)
  14. 洗涤缓冲液A(参见配方)
  15. 洗涤缓冲液B(见配方)
  16. 洗脱缓冲液(参见配方)
  17. 2x结合缓冲液(参见配方)

设备

  1. 培养管13ml(SARSTEDT,目录号:62.515.028)
  2. NanoDrop 2000c UV-Vis分光光度计(Thermo Fisher Scientific,目录号:ND-2000c)
  3. 高性能模块化立体显微镜,用于基于应用的定制MZ6(Leica Microsystems,型号:MZ6)
  4. 球磨机(RETSCH,型号:MM200)
  5. 热循环仪(Eppendorf,目录号:950000031)
  6. MiniSpin ?/MiniSpin ?加离心机(Eppendorf,目录号:022620100)
  7. MagneSphere Technology Magnetic Separation Stands(Promega Corporation,目录号:Z5342)

程序

  1. 冠状肿瘤诱导
    1. A的过夜文化。从生长的菌落获得根癌土壤杆菌 在KB琼脂平板上。将菌落转移到3-5ml KB液体中 培养基在具有组装的两位置通风的13ml培养管中 帽(位置1:盖轻轻推,通气)并孵育 在旋转振荡器中在140rpm下在28℃下过夜
    2. 隔夜。将根癌土细胞以8,000rpm离心1分钟 悬浮于3ml agromix缓冲液中。在28℃培养悬浮细胞 再次140rpm,保持2-3小时。 OD 600通过分光光度计测量 ?调节至OD 600 = 0.5。
    3. 年轻花序茎(3至10厘米) ?的拟南芥在底部附近用5ml注射器注射两次 次,在另一个垂直方向为两次 (图1)。注射器针被完全注射通过 茎。一滴 A。根瘤土细胞用于一次注射。


    图1.拟南芥膨胀茎的示意图。用注射器在注射区(标记)注射年轻的花序茎。原始的拟南芥图片来自网站(
    http://www.lookfordiagnosis.com/mesh_info.php?term=Arabidopsis&lang=1 )。

    1. 茎总共注射8次,因此通常8个块 ?的肿瘤在接种后25天产生(图2)。的 用手术刀和镊子在茎下从茎切下肿瘤 解剖显微镜Leica MZ6和称重在天平。


    图2. 拟南芥冠gall肿瘤。在接种后25天,从冠状动物的茎产生冠gall肿瘤。用手术刀和镊子切割肿瘤(用红色圆圈标记)并称重
  2. Poly-A-mRNA提取
    大约50mg冠gall肿瘤用于mRNA提取。通过与聚苯乙烯珠结合的寡核苷酸Poly-dTTP(25)分离聚-A-mRNA。使用Dynabeads Oligo- [dT] 25试剂盒
    1. 将植物组织在液氮中冷冻,并在室温下均化 球磨机,然后加入1ml裂解缓冲液,样品为 在室温下进一步匀浆1-2分钟,直到组织 ?完全裂解。将裂解物在10,000rpm离心30秒。
    2. Dynabeads Oligo- [dT] 25试剂盒的制造商方案 执行。将上清液与20μlDynabeads孵育30分钟 ?在室温下在顶置转子中。管子放在 MagneSphere ?技术磁性分离器放置2分钟并取出 ?上清液。磁性架的制造商方案是 ?执行。
    3. 第一洗涤步骤:洗涤Dynabeads 用200μl洗涤缓冲液A洗涤两次,并用200μl洗涤缓冲液洗涤两次 ?B.磁体用于分离珠子和上清液 洗涤。通过轻轻地冲洗Dynabeads的底部来洗涤 管,直到所有珠子重悬浮,然后捕获珠子 通过磁性架。除去上清液而不干扰 珠子
    4. 为了排除DNA污染,mRNA是 通过50μl洗脱缓冲液从珠子上洗脱并在75℃下孵育 2分钟。
    5. 第二个洗涤步骤:50μl2x结合缓冲液 加入到洗脱的mRNA中,再次温育100μl混合物 20分钟,在室温下在顶置式振荡器中。
    6.  两后 用200μl洗涤缓冲液B洗涤步骤,mRNA从洗脱液中洗脱 ?磁珠与22μl洗脱缓冲液在75℃下2分钟。的 通过NanoDrop 2000c UV-Vis定量mRNA的浓度 分光光度计。 mRNA直接用于第一链cDNA 合成用于5'RACE测定或储存在-80℃。
  3. 5'- cDNA末端快速扩增(5'RACE)测定
    通过SMARTer TM RACE cDNA扩增试剂盒扩增致癌基因cDNA的5'末端。第一链cDNA由SMARTScribe TM逆转录酶产生并使用SMARTer II A寡核苷酸和5'-CDS引物A引发。
    1. 将下列试剂加入一个管中
      2.75μl        mRNA
      1.0μl5'-CDS引物A
      通过移液管将试剂充分混合,并在72℃下孵育3分钟,然后在42℃下孵育2分钟。
    2. 将下列试剂加到来自步骤a的混合物中,总体积为10μl
      1.0μl         SMARTer II A寡核苷酸
      2.0μl         5x First-Strand Buffer
      1.0μl         DTT(20 mM)
      1.0μl         dNTP Mix(10 mM)
      0.25μl      核糖核酸酶抑制剂(40 U /μl)
      1.0μl         SMARTScribe?反转录酶(100 U)
      用移液管充分混合试剂,并在42℃下孵育试管90分钟
    3. 将反应混合物在70℃加热10分钟以终止反应 ?反应,并将第一链反应产物用10稀释 折叠ddH 2 O。将样品储存在-20℃或用于下一步骤 直。
  4. 癌基因cDNA 5'端的扩增和测序
    1. 通过扩增癌基因cDNA的5'末端的片段 DreamTaq DNA聚合酶,并使用通用引物A混合物(UPM) 和特异性靶引物(表1),如以下反应所示 混合物 PCR反应(总体积50μl):
      来自步骤3.3.1的cDNA                           1微升
      5x缓冲区                                                 10微升
      10x通用引物A混合                        5微升
      特异性靶反向引物(10μM)        1微升
      dNTP(10mM)                       ;                  1微升
      DreamTaq DNA聚合酶                 0.5μl
      ddH 2 O,最终卷为                ;    50微升
      PCR程序:
      步骤1:95°C                     1 min
      步骤2:95°C                    30秒
      步骤3:55°C                    30秒
      第4步:72°C                    15秒
      步骤5:重复步骤2-4 30个周期
      步骤6:72°C                     5分钟
      步骤7:4°C                      商店

    表1.特异性癌基因反向引物
    基因名(保藏号)
    引物序列(5'→3')
    IaaH (pTiC58,AE007871)
    CCCCGATTGCTAACAGACG
    IaaM (pTiC58,AE007871)
    CAAGAGTGTTCGAGAGG
    Ipt (pTiC58,AE007871)
    TCCCATGAATCAACTTAT

    1. PCR产物通过QIAquick PCR纯化试剂盒纯化
    2. 将纯化的PCR产物通过TA克隆插入pGEM-T Easy Vector 2x快速连接缓冲液                          5微升
      pGEM-T Easy Vector(50 ng /μl)         0.5μl
      PCR产品                                     3.5μl
      T4 DNA连接酶                                     1微升
      通过移液管混合试剂,并在室温下孵育1小时
    3. 将得自步骤4c的重组载体转化进E。使用热休克法的大肠杆菌菌株MRF'。
    4. 对5个以上的独立菌落进行5'端测序 PCR产物,其是癌基因的转录起始位点。

代表数据

表2.癌基因启动子中的 Cis - 调节序列元件

该数据在PLOS Pathogens上发表(Zhang等人,2015)。正数表示下游的位置,负数表示TSS上游的位置(+1)。 TSS下划线。 Y = C/T,W = A/T,N = A/G/C/T。 (Klee等人,1984); (Nester et al。,1984); (Goldberg等人,1984); 4.(Heidekamp等人,1983); (de Pater et al。,1987); (Lichtenstein ed。,et al。,1984)

食谱

  1. KB媒体
    20克蛋白酶蛋白胨 1.5克K sub 2 HPO 4
    0.87%甘油 600μMMgSO 4/v/v 15克琼脂(仅板)
    高压灭菌器灭菌
  2. Agromix缓冲区
    0.01M MgCl 2/
    0.01 M MES
    pH 5.6
  3. 裂解缓冲液
    100mM Tris-HCl(pH7.5) 500mM LiCl 10 mM EDTA
    1%LiDS
    5mM二硫苏糖醇(DTT) 如果观察到任何沉淀,将缓冲液加热至室温并摇动,直至所有组分完全重悬
  4. 洗涤缓冲液A
    10mM Tris-HCl(pH7.5) 0.15 M LiCl
    1mM EDTA
    0.1%LiDS
  5. 洗涤缓冲液B
    10mM Tris-HCl(pH7.5) 0.15 M LiCl
    1mM EDTA
  6. 洗脱缓冲液
    10mM Tris-HCl(pH7.5)
  7. 2x结合缓冲液
    20mM Tris-HCl(pH7.5) 1.0 M LiCl
    2mM EDTA

致谢

我要感谢Rosalia Deeken(德国维尔茨堡大学分子植物生理学和生物物理系)对这项工作的监督和中国奖学金委员会(CSC)的财政支持。我还要感谢德国维尔茨堡大学分子植物生理学和生物物理学系高强高,帮助,建议和分享试剂。

参考文献

  1. de Pater,B.S.,Klinkhamer,M.P.,Amesz,P.A.,de Kam,R.J.,Memelink,J.,Hoge,J.H.and Schilperoort,R.A。(1987)。 土壤杆菌T-cyt基因的植物表达信号。 Nucleic Acids Res 15(20):8267-8281。
  2. Goldberg,S.B.,Flick,J.S.and Rogers,S.G。(1984)。 根癌农杆菌的tmr基因座的核苷酸序列 pTi T37 T-DNA 。 Nucleic Acids Res 12(11):4665-4677。
  3. Heidekamp,F.,Dirkse,W.G.,Hille,J.and van Ormondt,H。(1983)。 根癌农杆菌章鱼碱Ti质粒的核苷酸序列编码d tmr基因。 Nucleic Acids Res 11(18):6211-6223。
  4. Klee,H.,Montoya,A.,Horodyski,F.,Lichtenstein,C.,Garfinkel,D.,Fuller,S.,Flores,C.,Peschon,J.,Nester,E。和Gordon, 1984)。 pTiA6NC章鱼碱Ti质粒的tms基因的核苷酸序列:涉及植物肿瘤??发生的两种基因产物。 Proc Natl Acad Sci USA 81(6):1728-1732。
  5. Lichtenstein,C.,Klee,H.,Montoya,A.,Garfinkel,D.,Fuller,S.,Flores,C.,Nester,E.and Gordon,M。(1984)。 pTiA6NC章鱼碱Ti质粒的tmr基因的核苷酸序列和转录物作图:涉及的细菌基因在植物肿瘤发生中。 2(4):354-362。
  6. Nester,E.W.,Gordon,M.P.,Amasino,R.M.and Yanofsky,M.F。(1984)。 冠gall - 分子和生理分析 Annual Review Of Plant Physiology And Plant Molecular Biology 35:387-413
  7. Zhang,Y.,Lee,C.W.,Wehner,N.,Imdahl,F.,Svetlana,V.,Weiste,C.,Droge-Laser,W.and Deeken, 在T-DNA转化的宿主植物细胞中调节癌基因表达。 PLoS Pathog 11(1):e1004620。
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Copyright: © 2017 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. Zhang, Y. (2015). 5’ Rapid Amplification of cDNA Ends (5’ RACE) of Agrobacterial T-DNA Genes within Transformed Plant Sample. Bio-protocol 5(18): e1603. DOI: 10.21769/BioProtoc.1603.
  2. Zhang, Y., Lee, C. W., Wehner, N., Imdahl, F., Svetlana, V., Weiste, C., Droge-Laser, W. and Deeken, R. (2015). Regulation of oncogene expression in T-DNA-transformed host plant cells. PLoS Pathog 11(1): e1004620.
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