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A Protocol to Determin Auxin Polar Transport Tate   

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实验原理:生长素极性运输在植物发育过程中发挥有非常重要的作用。在地上部分,生长素由顶端合成向基部运输,因此可以在茎杆顶端施加同位素H3标记的IAA,然后一定时间后检测另一端的同位素H3信号,从而可以评价生长素运输的速率。
实验目的:检测植物体内生长素极性运输的速率。

关键词: 生长素, 极性运输, 速率测定

材料与试剂

  1. 1.5 ml管子
  2. 滤纸
  3. 记号笔
  4. 水稻植株
  5. H3标记的IAA (Amersham, catalog number: TRK-781)
  6. Phytogel
  7. 生长素极性运输抑制剂TIBA (Sigma, catalog number: T5910)
  8. 液闪缓冲液 (见溶液配方)
  9. ½ MS液体培养基 (见溶液配方)
  10. ½ MS固体培养基 (见溶液配方)

仪器设备

  1. 液闪仪
  2. 摇床

实验步骤

  1. 取水稻抽穗期的第一节间,截取底端2 cm,每0.5 cm标记一次 (一定要严格区分顶端和底端),放入½ MS液体培养基(pH 5.8)中于摇床上, 100 rpm, 2 h以脱去内源的生长素。
  2. 用滤纸吸干后,将顶端和底端 (分别测量向基和向顶运输能力) 插入预先准备的20 μl含有100 nM的H3-IAA和0.35% phytogel的½ MS固体培养基中(1.5 ml管子),盖好盖子。
  3. 向基运输做两组,一组正常进行,另外一组,在½ MS固体培养基中还加入10 μM的TIBA。
  4. 上述处理材料在室温下暗处理3 h,然后从未插入培养基的另外一端截取0.5 cm长的茎秆片段,用½ MS液体培养基洗两次(15 min每次),放入2 ml液闪液中漂洗16~24 h,用液闪仪测量放射强度,按照参考文献中的计算方法将信号值转换为运输速度 (Lewis and Muday, 2009)。

注意事项

  1. 在脱去内源生长素的时候,要保证茎杆为垂直向上在摇床上慢摇。
  2. 所用材料不宜过老。
  3. 必须做向顶运输的对照,以排除检测到的信号值是因为IAA在茎杆中自由扩散而产生的。
  4. H3为辐射距离非常短的同位素类型,且释放的是高速运动电子,不会穿透人体,但是必须保证不能跟皮肤直接接触,否则会因为潜在的内照射而不安全。H3的半衰期长达12年以上,因此实验过程中产生的所有废料必须妥善的处置。

溶液配方

  1. ½ MS液体培养基,pH 5.8 
    先配制母液:
    1)
    MSmax solution
    NH4NO3
    16.5 g/L
    KNO3
    19.0 g/L
    KH2PO4
    1.7 g/L
    MgSO4·7H2O
    3.7 g/L
    CaCl2·2H2O
    4.4 g/L
    2)
    MSmin solution
    KI
    0.083 g/L
    H3BO3
    0.62 g/L
    MnSO4·4H2O
    2.23 g/L
    ZnSO4·7H2O
    0.86 g/L
    Na2MoO4·2H2O
    0.025 g/L
    CuSO4·5H2O
    0.0025 g/L
    CoCl2·6H2O
    0.0025 g/L
    3)
    ½ MS液体培养基配制
    取50 ml MSmax solution和5 ml MSmin solution,加入800 ml H2O,用1 M KOH调节pH到5.8,定容到1 L。
  2. ½ MS固体培养基
    在½ MS液体培养基中加入Phytagel 3.5 g/L加热融化后即得½ MS固体培养基。
  3. 液闪缓冲液
    (2,5-二苯基恶唑) (PPO) 5 g/L;1, 4-双-(4’-甲基-5-苯基恶唑) (POPOP) 0.2 g/L,用甲苯溶解。

参考文献

  1. Lewis, D. R. and Muday, G. K. (2009). Measurement of auxin transport in Arabidopsis thaliana. Nat Protoc 4(4): 437-451.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:王磊, 和玉兵, 欧阳亦聃. (2018). 生长素极性运输速率检测方法. Bio-101: e1010173. DOI: 10.21769/BioProtoc.1010173.
How to cite: Wang, L., He, Y. B. and Ouyang, Y. D. (2018). A Protocol to Determin Auxin Polar Transport Tate. Bio-101: e1010173. DOI: 10.21769/BioProtoc.1010173.
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