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Particle bombardment has been shown to be a useful method to study gene promoter regulatory elements by transient transformation of maize embryos with different constructions of gene promoters fused to a gene reporter. DNA to transfer is coated to high density gold microparticles and introduced into cells when accelerated by a helium pulse. This method allows a first rapid approach, avoiding time consuming stable transformation of maize plants and also allows quantitative promoter expression analysis by a histochemical or fluorometric assay.

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Maize Embryo Transient Transformation by Particle Bombardment
基因枪法介导玉米胚的瞬时转化

植物科学 > 植物转化 > 基因枪法
作者: Matilde Jose-Estanyol
Matilde Jose-EstanyolAffiliation: Centre de Recerca en Agrigenòmica (CRAG), CSIC-IRTA-UAB-UB, Edifici CRAG, Campus UAB, Bellaterra, Barcelona, Spain
For correspondence: matilde.jose@cragenomica.es
Bio-protocol author page: a768
Vol 3, Iss 16, 8/20/2013, 3211 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.865

[Abstract] Particle bombardment has been shown to be a useful method to study gene promoter regulatory elements by transient transformation of maize embryos with different constructions of gene promoters fused to a gene reporter. DNA to transfer is coated to high density gold microparticles and introduced into cells when accelerated by a helium pulse. This method allows a first rapid approach, avoiding time consuming stable transformation of maize plants and also allows quantitative promoter expression analysis by a histochemical or fluorometric assay.
Keywords: Particle bombardment(粒子轰击), Maize embryos(玉米胚), Promoter expression(启动子表达), Quantitative analysis(定量分析)

[Abstract]

Material and Reagents

  1. Plant Material: Maize plants from W64A pure inbred line
  2. Ethanol absolute, gradient HPLC grade (Scharlau, S.A. UN1170)
  3. 1.0 μm Gold Microcarriers (Bio-Rad Laboratories, catalog number: 165-2263 )
  4. Rupture Disks 900 psi (Bio-Rad Laboratories, catalog number: 165-2328 )
  5. Macrocarriers (Bio-Rad Laboratories, catalog number: 165-2335 )
  6. Stopping Screens (Bio-Rad Laboratories, catalog number: 165-2336 )
  7. Murashige and Skoog medium (M&S) with vitamins (Duchefa, catalog number: M0222 )
  8. Spermidine free base (Sigma-Aldrich, catalog number: S-2626 )
  9. Sodium hypochlorite, solution 5-9% Cl active (Carlo Erba, catalog number: CH0223 )
  10. Cell culture dishes 60 mm x 15 mm Style treated polystyrene (Corning Inc., catalog number: 430166 )
  11. X-GlcA sodium trihydrate (Duchefa, catalog number: X-1406 )
  12. X-glucuronide: 5 bromo-4-chloro-3-indolyl-β-D-glucoronic sodium salt 3H2O
  13. N,N-dimethylformamid for spectroscopy (Merck KGaA, catalog number: 102937 )
  14. MUG: 4 methyl-umbelliferyl-B-D-glucuronide hydrate C (Sigma-Aldrich, catalog number: M9130 )
  15. Potassium hexacyanoferrate (III) (Merck KGaA, catalog number: 104973 )
  16. Potassium hexacyanoferrate (II) trihydrate (Merck KGaA, catalog number: 104984 )
  17. 4-Methylumbelliferone sodium salt (4-MU) (Sigma-Aldrich, catalog number: M-1508 )
  18. Luciferase Assay Reagent (Promega Corporation, catalog number: E1500 )
  19. CDTA: trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, catalog number: D0922 )
  20. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  21. Histochemical analysis detection buffer (see Recipes)
  22. Fluorometric lysis buffer (see Recipes)
  23. Fluorometric analysis reaction buffer (see Recipes)
  24. Fluorimetric analysis stopping buffer (see Recipes)
  25. MSO medium (see Recipes)
  26. 1x Luciferase lysis buffer (see Recipes)

Equipment

  1. BIORAD PDS-1000/He system (Biolistic Delivery Systems)
  2. Laminar Flow Cabinet Telstar AH-100
  3. Vacuum pump Telstar 50/60 Hz
  4. Cylinder compressed helium gas (UN1046) 117 Kg (LYNDE)
  5. RAYPA Bathwater sonicator 50 W
  6. MINI-SECOEUR IKA MS2 minishaker with a support that allows simultaneous agitation of different Eppendorf tubs
  7. MiniSpin eppendorf centrifuge
  8. ND-1000 Spectrophotometer (NanoDrop)
  9. Spectra Max M3 apparatus (bioNova cientifica S.L.)
  10. Olimpus Stereomicroscope SZX16
  11. Screw cylinder polypropylene microtube with attached O-Ring cap and conical base (Sarstedt, catalog number: 72.693.105 )

Procedure

  1. Embryo excision from the maize kernel
    1. Submerge and wash maize ears 16-18 days after manual pollination (dap) successively in different solutions as follows at room temperature:
      1. 1 min with ethanol 96%. Discard.
      2. 10 min with 25% Sodium hypochlorite. Discard.
      3. 5 min sterile water (x 3 times). Discard.
    2. Detach grains with gloved hands from the washed ears and embryos from the grains with the help of a sterile cutter in a sterile Petri glass dish. Place 9 embryos/dish in a 3 x 3 array in the center of cell culture dishes (60 mm diameter) containing MSO medium. The embryo axis side in contact with the medium. Manipulations are done under a Laminar Flow Cabinet.
    3.  Maintain plates at 21-23 °C for 24 h in the dark before bombardment to allow embryos to be recovered of excision treatment. The described conditions have been shown not to alter the embryogenesis programme of the studied maize embryos (Jose-Estanyol et al., 2012).
  2. Microcarrier stock preparation (at room temperature)
    1. Add 1 ml ethanol (HPLC) to 60 mg of Gold Microcarriers in a cylinder microtube with attached ring cap and a conic base. Vortex at 2,200 rpm with the minishaker for 10 min. Avoid the use of hydrated ethanols.
    2. Centrifuge in the MiniSpin Eppendorf Centrifuge for 1 min at 10,000 rpm.
    3. Remove the Ethanol with a pipette, without removing microcarriers sediment. Discard ethanol.
    4. Add 1 ml sterile water. Vortex 1 min. Sediment by centrifugation as in step 2-b. Discard water (Repeat 3 times).
    5. Suspend microcarriers in 1 ml of water. Vortex 1 min. Make aliquots (30 μl) in conical tubes from the homogeneous suspension.
    6. Store at -20 °C.
  3. Microcarriers coating with the plasmid DNA of interest
    1. Quantify DNA stocks (prepared by using a commercial plasmid prep kit) of plasmids sharing the studied promoters fused to a gene reporter, usually the beta-glucoronidase enzyme, with a nanodrop Spectrophotometer.
    2. Sonicate defreezed microcarrier aliquots for 3 min in a bathwater sonicator.
    3. Add succesivelly to the microcarrier aliquots:
      12.5 μl DNA in TE buffer (1 μg/μl)
      95 μl of water
      125 μl CaCl2 2.5 M (while vortexing: Open the tube and decrease speed to avoid sample lost while you add solutions)
      25 μl spermidine 0.1 M (while vortexing)
    4. Vortex for 3-5 min.
    5. Allow sedimentation on ice to minimize ethanol evaporation for 15-20 min.
    6. Discard solution without disturbing microcarrier sediment as in step 2-c.
    7. Add 500 μl ethanol (HPLC). Vortex 10-20 sec. Allow sedimentation during 15-20 min on ice.
    8. Discard ethanol as in step 2-c.
    9. Repeat with 200 μl ethanol (HPLC).
    10. Discard ethanol as in step 2-c.
    11. Finally add 40 μl ethanol (HPLC).
    12. Sonicate in the bathwater sonicator 3 sec (Repeat 3 times).
    13. Vortex 3 min.
    14. Distribute the 40 μl homogenous solution of DNA coated microcarriers in ethanol (HPLC) (step 3-j) between the surface center of three macrocarriers (≈ 10 μl/macrocarrier) on a flat leveled surface. Macrocarriers have been washed in 70% ethanol and dried, beforehand. Let dry. This will allow the bombardment of three different samples (three cell culture dishes, each with 9 embryos in MSO medium).
    15. When microcarriers have dried on macrocarriers surface (5-10 min), they are introduced inside previously autoclaved macrocarriers holders and are ready to be used for maize embryo bombardment.
  4. Maize embryos 16-18 dap particle bombardment
    1. Wash stopping screens and rupture disks with 70% ethanol. Locate in the adaptor the stopping screen and the macrocarrier holder with microcarriers attached to the macrocarrier. Locate the rupture disk at the end of the acceleration tube.
    2. Proceed to the bombardement of the cell culture dishes with the maize embryos in MSO medium with the microcarriers coated with the different studied plasmids following PDS-100/He System instructions (http://www.Bio-Rad.com/biolostics).
    3. Bombardement parameters are as follows:
      Gap distance
      1.0 cm
      Macrocarrier travel distance
      1.5 cm
      Target distance
      9.5 cm
      Gold microcarriers
      1.0 μm
      Camera partial vacuum
      0.1 atm
      Rupture Disks
      900 psi
      Helium pressure at the regulator
      1,100 psi
    4. After bombardment and before the quantitative fluorometric or histochemical analysis, Petri dishes are incubated in the dark at 21-23 °C for 24 h to allow expression of the reporter gene. Fluorometric assay is very useful for strong promoters but for low or medium ones, the histochemical analysis can be more sensitive and avoid dilution of the signal (present mainly in the scutellum first cell layer of the bombarded embryos, penetration depth). In histochemical analysis better quantification can be achieved when both the number and the diameter of blue spots (see Figure 1) are quantified (for example: twelve basic units for spots with 80 μm diameter, six basic units for spots with 40 μm diameter, 3 basic units for spots with 20 μm diameter and one basic unit for spots with less than 20 μm diameter).
    5. The validity of results is evaluated by the reproducibility of the results. This is achieved by the mean value of different experiments (3 to 4) and their standard deviation.
    6. Finally significant differences between constructs or conditions (p-value) are calculated from the means of different experiments in a Student’st test (http://www.physics.csbsju.edu).
    7. Different efficiency has been observed in different maize varieties. Efficiency has shown to be in an inverse rapport with the scutellar hydration level degree of the different maize varieties during embryo development.
  5. Quantitative histochemical analysis
    1. Embryos from each bombarded dish are transferred to 2 ml eppendorf tubes with 1 ml of histochemical analysis detection buffer and incubated overnight at 37 °C.
    2. Blue spots of different intensity appear as result of the precipitation of product derivates reaction, see Figures 1-2 (This reaction is greatly enhanced by using an oxidation catalyst such as a potassium ferricyanide/ferrocyanide mixture).
    3. To avoid diffusion of the blue spots on the surface of the embryos, they are transferred to a 50% glycerol solution in water. Blue spots are quantified by observation with an OLYMPUS Stereomicroscope SZX16. Stained embryos are stored at 4 °C.
    4. As control one set of embryos can be bombarded with a constitutive promoter for monocots, such as OsActine: GUS-nos ter, to easily evaluate the efficiency of the microcarrier batch and of each specific experiment.
    5. Histochemical analysis examples:


      Figure 1. Maize embryos bombarded with a promoter with a medium-low expression level


      Figure 2. Set of maize embryos bombarded with OsActine::Gus-nos ter constitutive strong promoter

  6. Quantitative fluorometric analysis
    1. Embryos from each bombarded dish are frozen in liquid N2 and stored at -80 °C until analysis.
    2. The nine frozen embryos from a bombarded dish are grinded in 300 μl fluorometric lysis buffer.
    3. Centrifuge 15 min 13,000 rpm in the MiniSpin Eppendorf Centrifuge at 4 °C.
    4. Freeze the clear extract solution at -80 °C in aliquots of 40 μl, until beta-glucoronidase quantification. Sediment is rejected.
    5. For fluorometric analysis 20 μl of clean extract are added to 80 μl fluorometric analysis reaction buffer and warmed at 37 °C.
    6. Aliquots of 20 μl are taken at different time courses (10 (zero), 60, 180, 360 min).
    7. Reaction is stopped by an addition of 180 μl of fluorimetric analysis stopping buffer to each taken aliquot and stored 4 °C until analysis.
    8. Samples are transferred to a microtiter plate to measure fluorescence emission of the beta-glucoronidase enzyme product 4-MU (4-methylumbelliferone) in a plate fluorescence lector, Spectra Max M3 apparatu (excitation 365 nm, emission 455 nm).
    9. After analysis defreezed samples can not be refreezed and are discarded.
    10. 4-Methylumbelliferone (4-MU) standards are used to calibrate the system.
    11. Protein concentration in the extracts is measured using the Bradford assay in a Spectra Max M3 apparatus.
  7. Internal control in fluorometric studies, Two different internal controls are suggested
    1. Samples are cobombarded with a construction of luciferase enzyme fused to a constitutive promoter as maize ubiquitin (pUBI::LUC-nos-ter) to control homogenous bombardment of the different samples in an experiment.
      1. Freeze the embryos in liquid nitrogen after 24 h of bombardment, grind all the frozen tissue to a powder and resuspend by homogenization in 300 μl Luciferase lysis buffer (1x) previously tempered to room temperature.
      2. Sediment debris by brief centrifugation at 13,000 rpm with the MiniSpin Eppendorf Centrifuge at 4 °C and transfer supernatant to a new tube. Maintain on ice until analysis.
      3. Mix 10 μl of cell lysate with 100 μl of Luciferase Assay Reagent in a microtiter plate and quickly measure the light produced in a Spectra Max M3 apparatus.
      4. Make 40 μl aliquots with the remaining extract and freeze at -80 °C. Then proceed from step 6-e to do the fluorometric analysis of the studied promoters.
    2. Alternatively samples can be cobombarded with a mixture of two constructions that express two maize complementary transcriptional factors, the maize myb factor C1 (35S::I-C1) and the myc factor B-Peru (35S::I-B-Peru) involved in the anthocyanin biosynthesis. In these constructions the expression of these factors is under the control of the cauliflower mosaic virus (CAMV) 35S constitutive promoter and the first intron enhancer of the maize Adh1 (alcohol dehydrogenase 1). Expression of both factors results in red/bronze spots on the surface of the embryos that can be visually compared by observation with a stereomicroscope. Then proceed from step 6-a to do the fluorometric analysis of the studied promoters.

Recipes

  1. Histochemical analysis detection buffer (10 ml)
    1 ml sodium phosphate 1 M (pH 8.0)
    27 mg (final concentration, 5 mM) Potassium hexacyanoferrate (III)
    21 mg (final concentration, 5 mM) Potassium hexacyanoferrate (II)
    6 μl Triton X-100
    1.5 ml X-Gluc from stock (20 mg/ml in N,N-dimethylformamid)
  2. Fluorometric lysis buffer
    50 mM sodium phosphate (pH 7.0)
    10 mM EDTA (pH 8.0)
    10 mM BME (beta-Mercaptoethanol)
    0.1% SDS (v/v)
    0.1% Triton X-100 (v/v)
  3. Fluorometric analysis reaction buffer (4 ml)
    3 ml lysis buffer
    1 ml methanol
    1,764 mg MUG
  4. Fluorimetric analysis stopping buffer
    0.2 M Na2CO3
  5. MSO medium(1 L)
    4.5 g M&S medium with vitamins (pH 5.8 with KOH 1 M)
    30 g sucrose
    2.4 g Gelrite
  6. 1x Luciferase lysis buffer
    25 mM Tris-phosphate (pH 7.8)
    2 mM DTT
    2 mM CDTA:1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid
    10% glycerol
    1% Triton® X-100

Acknowledgments

This protocol is adapted from Jose-Estanyol and Puigdomenech (2012).

References

  1. Jose-Estanyol, M. and Puigdomenech, P. (2012). Cellular localization of the embryo-specific hybrid PRP from Zea mays, and characterization of promoter regulatory elements of its gene. Plant Mol Biol 80(3): 325-335.

材料和试剂

  1. 植物材料:来自W64A纯近交系的玉米植物
  2. 乙醇绝对梯度HPLC级(Scharlau,S.A.UN1170)
  3. 1.0微米金微载体(Bio-Rad Laboratories,目录号:165-2263)
  4. 破裂盘900psi(Bio-Rad Laboratories,目录号:165-2328)
  5. 大载体(Bio-Rad Laboratories,目录号:165-2335)
  6. 停止筛(Bio-Rad Laboratories,目录号:165-2336)
  7. 具有维生素(Duchefa,目录号:M0222)的Murashige和Skoog培养基(M& S)
  8. 亚精胺游离碱(Sigma-Aldrich,目录号:S-2626)
  9. 次氯酸钠,5-9%Cl活性溶液(Carlo Erba,目录号:CH0223)
  10. 细胞培养皿60mm×15mm样式处理的聚苯乙烯(Corning Inc.,目录号:430166)
  11. X-GlcA三水合钠(Duchefa,目录号:X-1406)
  12. X-葡萄糖醛酸苷:5-溴-4-氯-3-吲哚基-β-D-葡糖醛酸钠盐3H 2 O
  13. 用于光谱的N,N-二甲基甲酰胺(Merck KGaA,目录号:102937)
  14. MUG:4甲基伞形基-β-D-葡萄糖醛酸苷水合物C(Sigma-Aldrich,目录号:M9130)
  15. 六氰基铁酸钾(III)(Merck KGaA,目录号:104973)
  16. 六水合氰酸铁(II)三水合物(Merck KGaA,目录号:104984)
  17. 4-甲基伞形酮钠盐(4-MU)(Sigma-Aldrich,目录号:M-1508)
  18. 荧光素酶测定试剂(Promega Corporation,目录号:E1500)
  19. CDTA:反式-1,2-二氨基环己烷-N,N,N',N'-四乙酸(Sigma-Aldrich,目录号:D0922)
  20. 蛋白质测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  21. 组织化学分析检测缓冲液(参见配方)
  22. 荧光裂解缓冲液(参见配方)
  23. 荧光分析反应缓冲液(参见配方)
  24. 荧光分析停止缓冲液(参见配方)
  25. MSO介质(参见配方)
  26. 1x荧光素酶裂解缓冲液(参见配方)

设备

  1. BIORAD PDS-1000/He系统(Biolistic Delivery Systems)
  2. 层流柜Telstar AH-100
  3. 真空泵Telstar 50/60 Hz
  4. 气缸压缩氦气(UN1046)117 Kg(LYNDE)
  5. RAYPA浴水超声仪50 W
  6. MINI-SECOEUR IKA MS2小型支架,可同时搅拌不同的Eppendorf管子
  7. MiniSpin eppendorf离心机
  8. ND-1000分光光度计(NanoDrop)
  9. Spectra Max M3设备(bioNova cientifica S.L.)
  10. Olimpus立体显微镜SZX16
  11. 附带O型圈帽和锥形底座的螺旋圆柱聚丙烯微型管(Sarstedt,目录号:72.693.105)

程序

  1. 胚胎切除从玉米核
    1. 淹没   并在手工授粉后16-18天洗涤玉米穗(dap) 在不同的溶液中如下在室温下连续:
      1. 用乙醇96%洗脱1分钟。 舍弃。
      2. 用25%次氯酸钠洗涤10分钟。 舍弃。
      3. 5分钟无菌水(×3次)。 舍弃。
    2. 分离   谷物用手套的手从被洗涤的耳朵和胚胎从 谷物在无菌培养皿玻璃皿中的无菌切割器的帮助下。 将9个胚胎/培养皿置于细胞培养中心的3×3阵列中 (60mm直径),含有MSO培养基。 胚轴侧 接触介质。 操作在层流下进行 内阁。
    3.  在黑暗中将培养皿在21-23°C保持24小时   在轰击之前允许胚胎被恢复切除 治疗。 所描述的条件已经显示不改变 研究的玉米胚胎的胚胎发生程序(Jose-Estanyol等人,2012)。
  2. 微载体原液制备(在室温下)
    1. 加   1ml乙醇(HPLC)至60mg的金微载体 连接环帽和锥形底座的微管。 以2200rpm旋转 用minishaker 10分钟。 避免使用水合物 乙醇。
    2. 在MiniSpin Eppendorf离心机中以10,000rpm离心1分钟
    3. 用移液管除去乙醇,不除去微载体沉积物。 弃去乙醇。
    4. 加入1ml无菌水。 涡旋1分钟。 如步骤2-b中的离心沉淀。 弃去水(重复3次)。
    5. 将微载体悬浮在1ml水中。 涡旋1分钟。 从均匀悬液中取等分试样(30μl)在锥形管中
    6. 储存于-20°C。
  3. 用感兴趣的质粒DNA包被的微载体
    1. 量化   DNA储备液(通过使用商业质粒制备试剂盒制备) 通常将共享研究的启动子的质粒与基因报道子融合   β-葡萄糖醛酸酶,用纳米分光光度计。
    2. 超声处理解冻的微载体等分3分钟在浴水超声波仪
    3. 成功添加到微载体等分试样:
      12.5μl在TE缓冲液中的DNA(1μg/μl)
      95微升水
      125μlCaCl 2 2.5M(同时涡旋:打开管并降低速度以避免样品在添加溶液时丢失)
      25微升亚精胺0.1M(同时涡旋)
    4. 涡旋3-5分钟。
    5. 允许在冰上沉淀,以使乙醇蒸发最少15-20分钟
    6. 如步骤2-c所述,丢弃溶液而不干扰微载体沉淀
    7. 加入500μl乙醇(HPLC)。 涡旋10-20秒。 允许在冰上沉降15-20分钟。
    8. 如步骤2-c中那样丢弃乙醇。
    9. 用200μl乙醇(HPLC)重复。
    10. 如步骤2-c中那样丢弃乙醇。
    11. 最后加入40μl乙醇(HPLC)
    12. 在浴水超声仪中超声3秒(重复3次)。
    13. 涡旋3分钟。
    14. 分发   40微升DNA包被的微载体在乙醇中的均匀溶液 (HPLC)(步骤3-j)在三个大载体的表面中心之间(≈ 10微升/大载体)在平坦的水平表面上。 大载体已经 在70%乙醇中洗涤并预先干燥。 让干。 这将允许 对三种不同样品(三种细胞培养皿, 每个在MSO培养基中有9个胚胎)
    15. 当微载体有    在大载体表面(5-10分钟)上干燥,引入它们 内部以前高压灭菌的宏载体持有人和准备好 用于玉米胚胎轰击
  4. 玉米胚16-18 dap粒子轰击
    1. 洗   用70%乙醇终止筛和破裂盘。 位于 适配器的停止屏和宏载体托架 微载体附着于大载体。 找到爆破片 加速管的末端。
    2. 继续 用MSO中的玉米胚胎轰击细胞培养皿 培养基中包被有不同研究的质粒的微载体    遵循PDS-100/He系统说明( http://www.Bio-Rad.com/biolostics)。
    3. Bombardement参数如下:
      间隙距离
      1.0厘米
      宏载波旅行距离
      1.5厘米
      目标距离
      9.5厘米
      金微载体
      1.0微米
      相机部分真空
      0.1 atm
      破裂盘
      900 psi
      调压器氦气压力
      1,100 psi
    4. 后   轰击和在定量荧光或组织化学之前 分析,将培养皿在黑暗中在21-23℃孵育24小时 h以允许报告基因的表达。 荧光测定法 非常有用的强启动子,但对于低或中等, 组织化学分析可以更灵敏和避免稀释 信号(主要存在于盾片第一细胞层中 轰击胚,穿透深度)。 在组织化学分析更好 量化可以实现当数量和直径两者 蓝色斑点(参见图1)被量化(例如:十二基本的 单位为直径80μm的点,六个基本单位为40 μm直径,3个基本单位用于20μm直径和一个基本的点 单位用于直径小于20μm的点)。
    5. 的 结果的有效性通过结果的再现性进行评价。 这通过不同实验(3至4)和的平均值来实现   它们的标准偏差
    6. 最后有显着差异 在构建体或条件之间(

      http://www.physics.csbsju.edu ) 。

    7. 不同 在不同的玉米品种中观察到效率。 效率 已显示与盾片水合水平的逆相关   胚胎发育过程中不同玉米品种的程度
  5. 定量组织化学分析
    1. 胚胎   从每个轰击的皿转移到具有1的2ml eppendorf管   ml组织化学分析检测缓冲液并温育过夜 在37℃。
    2. 不同强度的蓝色斑点显示为 产物衍生物反应的沉淀结果,见图 1-2(通过使用氧化催化剂,该反应大大增强 例如铁氰化钾/亚铁氰化钾混合物)
    3. 至 避免蓝色斑点扩散到胚胎表面,他们 转移到50%甘油水溶液中。 蓝点是 通过用OLYMPUS立体显微镜SZX16观察来定量。 染色的胚胎在4℃贮存
    4. 作为控制一套 胚胎可以用单子叶植物的组成型启动子轰击,例如   作为 OsActine:GUS-nos ter ,以便轻松评估其效率 微载体批次和每个具体实验
    5. 组织化学分析实例:


      图1.用具有中低表达水平的启动子轰击的玉米胚胎


      图2.用OsActine :: Gus-nos 组成型强启动子轰击的玉米胚胎组 >
  6. 定量荧光分析
    1. 将来自每个轰击的皿的胚胎在液体N 2中冷冻并储存在-80℃直至分析。
    2. 将来自轰击皿的9个冷冻胚胎在300μl荧光裂解缓冲液中研磨
    3. 在MiniSpin Eppendorf离心机中在4℃下离心15分钟13,000rpm
    4. 冻结   澄清的提取物溶液在-80℃下等分40μl,直到 β-葡萄糖醛酸酶定量。 泥沙被拒绝。
    5. 对于荧光分析,将20μl纯净的提取物加入到80μl荧光分析反应缓冲液中并在37℃温热。
    6. 在不同的时间进程(10(零),60,180,360分钟)取20μl的等分试样
    7. 反应   通过加入180μl荧光测定分析终止 缓冲液加入到每份取出的等分试样中,并保存在4℃直至分析
    8. 样品 转移到微量滴定板以测量荧光发射 的β-葡萄糖醛酸苷酶产物4-MU(4-甲基伞形酮)   板荧光分析仪,Spectra Max M3 apparatu(激发365nm,发射455nm)
    9. 分析后,解冻样品不能重新冷冻并丢弃
    10. 4-甲基伞形酮(4-MU)标准品用于校准系统
    11. 蛋白   使用Bradford测定法测定提取物中的浓度   Spectra Max M3设备。
  7. 荧光测定研究中的内部控制,建议两个不同的内部控制
    1. 样品   被融合到a的荧光素酶酶的构建 组成型启动子作为玉米泛素(pUBI :: LUC-nos-ter)来控制 在实验中不同样品的同质轰击。
      1. 冻结   胚胎在液氮中24小时轰击后,研磨所有   冷冻组织粉末并通过匀浆在300μl重悬 将荧光素酶裂解缓冲液(1x)预先温热至室温
      2. 沉淀   通过用MiniSpin在13,000rpm下短暂离心来产生碎片 Eppendorf离心机在4°C,并将上清液转移到新管。 在冰上保持分析。
      3. 混合10微升细胞裂解液 用100μl的荧光素酶测定试剂在微量滴定板和 快速测量Spectra Max M3设备中产生的光。
      4. 使40μl等分试样 剩余的提取物并在-80℃下冷冻。 然后从步骤6-e进行到 进行研究的启动子的荧光分析
    2. 或者,可以用两种混合物来轰击样品 表达两种玉米互补转录的构建体 因子,玉米 myb因子C1 (<35> :: I-C1 )和 myc因子B-秘鲁 IB-Peru)参与花青素生物合成。 在这些 构造这些因素的表达受到控制 花椰菜花叶病毒(CAMV)35S组成型启动子和 第一个内含子增强子的玉米Adh1(醇脱氢酶1 )。 两种因子的表达导致表面上的红色/青铜斑点   可以通过观察用肉眼观察比较的胚胎 立体显微镜。 然后从步骤6-a开始进行荧光测定 研究的启动子的分析。

食谱

  1. 组织化学分析检测缓冲液(10ml)
    1ml磷酸钠1M(pH8.0) 27mg(终浓度5mM)六氰基铁酸钾(III)
    21mg(终浓度5mM)六氰基铁酸(II)钠 6微升Triton X-100 1.5ml来自储备液的X-Gluc(在N,N-二甲基甲酰胺中为20mg/ml)
  2. 荧光裂解缓冲液
    50mM磷酸钠(pH7.0) 10mM EDTA(pH8.0) 10mM BME(β-巯基乙醇) 0.1%SDS(v/v)
    0.1%Triton X-100(v/v)
  3. 荧光分析反应缓冲液(4ml)
    3ml裂解缓冲液
    1 ml甲醇
    1,764 mg MUG
  4. 停止缓冲液的荧光分析
    0.2M Na 2 CO 3 sub。
  5. MSO培养基(1L)
    4.5g具有维生素(pH 5.8,KOH 1M)的M& S培养基 30克蔗糖 2.4克Gelrite
  6. 1x荧光素酶裂解缓冲液
    25mM Tris-磷酸(pH7.8)
    2mM DTT
    2mM CDTA:1,2-二氨基环己烷-N,N,N',N'-四乙酸
    10%甘油 1%Triton X-100

致谢

该协议改编自Jose-Estanyol和Puigdomenech(2012)。

参考文献

  1. Jose-Estanyol,   M.和Puigdomenech,P。(2012)。 细胞定位 来自Zea mays的胚胎特异性杂交PRP,和表征 启动子调节元件的基因。植物分子生物学(Plant Mol Biol)80(3):325-335。
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How to cite this protocol: Jose-Estanyol, M. (2013). Maize Embryo Transient Transformation by Particle Bombardment. Bio-protocol 3(16): e865. DOI: 10.21769/BioProtoc.865; Full Text



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