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Determination of Primary Metabolites in Citrus Fruits   

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实验原理:初生代谢物是衡量柑橘果实品质及抗性的重要指标,通过GC-MS可精准高效的检测柑橘不同组织初生代谢物含量。
实验目的:测定柑橘果实各组织初生代谢物含量。

关键词: 柑橘, 果实, 初生代谢物

材料与试剂

  1. 1.5 ml离心管
  2. 10 ml离心管
  3. 1 ml注射器
  4. 0.22 µm微孔滤膜
  5. 气相质谱上样瓶
  6. 柑橘果实组织样品
  7. 液氮
  8. 甲醇
  9. 核糖醇
  10. 盐酸甲氧胺
  11. 吡啶
  12. BSTFA: (三甲基硅烷基)三氟乙酰胺N,O-bis (trimethylsilyl) trifluoroacetamide 
  13. TMCS: Trimethylchlorosilane
  14. 核糖醇内标 (见溶液配方)
  15. 盐酸甲氧胺(见溶液配方)

仪器设备

  1. 研钵
  2. -80 °C冰箱
  3. -20 °C冰箱
  4. 超声清洗仪
  5. 水浴锅
  6. 低温离心机
  7. 真空浓缩仪
  8. 真空干燥箱
  9. 气相色谱-质谱联用仪 (Thermo, model: DSQⅡ)

实验步骤

  1. 在研钵中充分研磨保存在-80 °C冰箱保存的柑橘果实样品,取150 mg充分研磨的样品加入盛有2,700 μl -20 °C预冷的甲醇的10 ml离心管中,摇匀后加入300 μl的核糖醇 (0.2 mg/ml) 内标,充分震荡至匀浆。
  2. 将样品置于超声清洗仪中4°C超声处理15 min,70°C水浴孵育15 min,置于-20°C冰箱使溶剂冷凝。
  3. 4°C,5,000 x g离心15 min;吸取100 µl上清于1.5 ml离心管中 (可以备份一份),30 °C真空浓缩至干后置于-80 °C超低温冰箱备用。
  4. 样品衍生化:在浓缩干的样品中加入50 μl的盐酸甲氧胺 (浓度为2 mg/ml,溶解于吡啶),50 °C于真空干燥箱孵育30 min,然后再加入50 μl BSTFA+1% TMCS于真空干燥箱中,60 °C孵育40 min。过0.22 µm微孔滤膜后,置于气相质谱上样瓶中以待上样。
  5. 气相色谱-质谱联用GC/MS (Thermo, DSQⅡ) 仪器运行条件:采用DB-5MS毛细管柱 (5%苯基/甲基聚硅氧烷,30 m x 0.25 mm x 0.25 μm),离子源 (70 eV),扫描范围为45-600 m/z,进样口温度为230 °C,传输线温度为250 °C,载气为高纯氦气 (99.999%),载气流量为1.0 ml/min;程序升温以100 °C开始,保持1 min,以3 °C/min升至184 °C,0.5°C /min升至190 °C保持1 min,15 °C/min升至280 °C保持5 min;分流比为10:1;手动上样,进样量为1 μl。

注意事项

  1. 组织样品一定要研磨充分。
  2. 上样前一定要过0.22 µm微孔滤膜,以防堵塞毛细管柱。

溶液配方

  1. 核糖醇内标:浓度为0.2 mg/ml,溶剂为蒸馏水
  2. 盐酸甲氧胺:浓度为2 mg/ml,溶剂为吡啶
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:朱峰, 许让伟, 刘淑桢, 程运江. (2018). 柑橘果实初生代谢物测定. Bio-101: e1010209. DOI: 10.21769/BioProtoc.1010209.
How to cite: Zhu, F., Xu, R. W., Liu, S. Z. and Cheng, Y. J. (2018). Determination of Primary Metabolites in Citrus Fruits. Bio-101: e1010209. DOI: 10.21769/BioProtoc.1010209.
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