一、视频摘要
本实验方法为快速酒精不溶性残留物制备法，首先用冰酒精使样品中的酶失活和溶解色素、其次多次使用酒精和丙酮完全除色素, 氯仿/甲醇除脂质，之后为了尽快干燥用甲醇代替之前溶剂，最终获得呈蓬松的白色/灰白色粉末状的细胞壁提取物。
This
method is rapid alcohol-insoluble residue preparation method. First,
use the cooled-alcohol to dissolve the pigment and inactive the
enzymes in samples. Second, use alcohol and acetone for many times to
completely remove the pigment, and use chloroform/methanol to remove
the lignin. Then, use methanol to replace the previous solvent in
order to dry as soon as possible. Finally, obtain the puffy
white/gray white powder cell wall extract.
二、关键词
丙酮、快速乙醇不溶性残留物制备法、叶片胞壁
Acetone,
Rapid alcohol-insoluble residue (AIR) preparation method, Leaf cell
wall
三、实验样品信息，试剂、耗材或仪器
1. 样品信息
青花菜叶片
Broccoli
leaf
2. 试剂和耗材
试剂：80%乙醇、丙酮、氯仿/甲醇混合液（1:1,v/v）、甲醇
耗材：2ml离心管
Reagent:
80% ethanol, acetone, chloroform/methanol mixture (1:1, v/v), ethanol
Consumables:
2ml centrifuge tube
3. 仪器和软件
移液枪、离心机、通风橱、冰箱、制冰机
Pipette,
Centrifuge, Laboratory chemical hood, Low-temperature refrigerator,
Ice machine.
四、实验操作
1.尽量避免使用锥底离心管进行实验，因为不利于涡旋。
2.80%乙醇需提前预冷。
3.乙醇浓度可在70-95%范围内进行调整，样品色素含量较高，建议使用较高的乙醇浓度。
4.胞壁提取物最后的干燥方式也可为：37~40℃烘干至恒重。
1.Try
to avoid using a cone bottom centrifuge tube for experiments, because
it makes subsequent vortex difficult.
2.80%
ethanol shall be precooled.
3.The
ethanol concentration can be adjusted within the range of 70-95%. The
pigment content of the sample is high, so it is recommended to use a
higher ethanol concentration.
4.
The drying method of the extract also be dried to constant weight at
37~40 ℃
五、注意事项
1. 因实验中存在大量有机溶剂、实验前请检查通风橱是否正常运转。
2. 确认实验手套是否耐受丙酮、氯仿、甲醇。
3. 植物组织注意充分研磨，过粗颗粒不利于有机溶剂去除色素等非胞壁物质。
1.
Since there are a lot of organic solvents in the experiment, please
check whether the Laboratory chemical hood operates normally before
the experiment.
2.
Confirm whether the test gloves are resistant to acetone, chloroform
and methanol.
3.The
plant tissue shall be fully ground, and the coarse particles are
unfavorable to the removal of pigment and other non-cell wall
substances by organic solvent.
六、结果分析
最终的细胞壁提取物呈蓬松的白色/灰白色粉末状。
The
final cell wall extract was fluffy white/grayish white powder.
七、参考文献
-
Pettolino
FA, Walsh C, Fincher GB and Bacic A, Determining the polysaccharide
composition of plant cell walls. Nat Protoc 7:1590-1607 (2012).
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Deng,
X., et al., Flag leaf cell wall functional groups and components
play a crucial role in the accumulation and translocation of Cd in
rice grain via foliage application of humic acid. Ecotoxicology and
Environmental Safety, 2022. 239: p. 113658.
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Hu
H, Brown PH and Labavitch JM, Species variability in boron
requirement is correlated with cell wall pectin. J Exp Bot
47:227-232 (1996).
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Luo
J, Ni D, Li C, et al. The relationship between fluoride accumulation
in tea plant and changes in leaf cell wall structure and composition
under different fluoride conditions[J]. Environmental Pollution,
2021, 270: 116283.
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Figueroa
C R, Opazo M C, Vera P, et al. Effect of postharvest treatment of
calcium and auxin on cell wall composition and expression of cell
wall-modifying genes in the Chilean strawberry (Fragaria chiloensis)
fruit. Food Chemistry [J], 2012. 132: 2014-2022.
-
黄伟男, 枇杷果实采后细胞木质素积累与细胞壁果胶动力学机制研究,
2021, 浙江大学.
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