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A method for efficiently extracting the inner mucus layer of Arabidopsis thaliana

A technology for Arabidopsis thaliana and mucus is applied in the field of efficient extraction of the inner mucus layer of Arabidopsis thaliana, which can solve problems such as difficulty in extracting the inner mucus layer, and achieve the effects of simplifying labor, reducing costs, and high extraction efficiency

Inactive Publication Date: 2016-12-21
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0004] The outer mucus layer of Arabidopsis thaliana is very easy to obtain. Just immerse the Arabidopsis seeds in water and shake for a few minutes, but the inner mucus layer is very difficult to extract. In scientific research experiments, the extraction of the inner mucus layer of Arabidopsis thaliana Mainly extracted by shaking, chemical reagents such as acid-base salts (Macquet A, Ralet MC, Kronenberger J, Marion-Poll A, North HM (2007) In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucosa. physiology 48:984-999), but these methods only extract a part of the inner mucus layer, and a large amount of inner mucus layer components remain on the seed coat

Method used

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  • A method for efficiently extracting the inner mucus layer of Arabidopsis thaliana
  • A method for efficiently extracting the inner mucus layer of Arabidopsis thaliana
  • A method for efficiently extracting the inner mucus layer of Arabidopsis thaliana

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Research on the extraction efficiency of Arabidopsis mucus layer by different extraction methods

[0055] (1) Preparation of extraction reagents: Prepare the following extraction reagents with purified water: 0.05M EDTA, 0.2% ammonium oxalate (w / v), 0.01M NaOH, 0.05M HCl.

[0056] (2) Extraction of Arabidopsis mucus layer with different chemical extraction reagents: Accurately weigh 5mg of Arabidopsis seeds, add 1mL of pure water, shake on a shaker at 150rpm for 5min, centrifuge at 3000rpm, take the supernatant as the outer layer of mucus Floor. Wash the seeds 3 times with purified water, add 1mL of corresponding reagents: purified water, 0.05M EDTA, 0.2% ammonium oxalate (w / v), 0.01M NaOH, 0.05M HCl, shake at 200rpm on a shaker at 40°C for 1h, centrifuge at 3000rpm, Take the supernatant, which is the inner mucus layer.

[0057] (3) Extraction of Arabidopsis mucus layer by ultrasonic treatment: Accurately weigh 5 mg of Arabidopsis seeds, add 1 mL of pure wa...

Embodiment 2

[0060] Example 2: Ultrasonic extraction of mucus layer of Arabidopsis thaliana

[0061] (1) Extraction of the outer mucus layer: Accurately weigh 5 mg of Arabidopsis seeds, add 1 mL of pure water, shake on a shaker at 150 rpm for 5 minutes, centrifuge at 3000 rpm, and take the supernatant to form the outer mucus layer.

[0062] (2) The same tube of seed samples undergoes mucus layer extraction at different ultrasonic stages: for the seeds in step (1), after cumulative ultrasonication of 5S, 10S, 20S, 40S, 1min, 2min, 4min, and 6min, the supernatants were taken respectively as internal layer mucus layer, and wash the seeds with purified water twice between each ultrasonic interval.

[0063] (3) Changes in the mucus layer of the same tube of seed samples after different ultrasonic stages: for the seeds in step (1), after accumulating ultrasonic waves of 0S, 5S, 10S, 20S, 40S, 1min, 2min, 4min, and 6min, the seeds were taken respectively, and each Wash the seeds with purified wa...

Embodiment 3

[0066] Example 3: Fluorescence Observation of Arabidopsis Seed Mucus Layer

[0067] (1) Arabidopsis seeds were shaken with water for 5 minutes to remove the outer mucus layer, then washed twice with 0.1M phosphate buffer solution (PhosphateBuffer Solution, PBS; 1L: 0.218g KH2PO4, 1.463g K2HPO4, 29.22g NaCl, pH 7.2) , 5min each time;

[0068] (2) Soak the seeds with fresh 5% (w / v) skimmed milk, shake on a slow shaker for 1 hour; remove the skimmed milk, and wash the seeds with PBS buffer for 5 minutes;

[0069] (3) Primary antibody incubation: Incubate the seeds with 20-fold diluted mouse anti-RG I antibody CCRC-M14 (Complex Carbohydrate Research Center) for 1 hour, and wash the seeds with PBS buffer 5 times to wash away the unbound primary antibody;

[0070] (4) Secondary antibody incubation: incubate with 50-fold diluted FITC-goat anti-mouse antibody (Comfort Century, Cat. No. CW0113S) for 1 h, and wash the seeds with PBS buffer 5 times to wash away the unbound secondary ant...

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Abstract

The invention discloses a method for efficiently extracting internal mucus layers of arabidopsis thaliana, belonging to the field of polysaccharide extraction. In the method disclosed by the invention, the internal mucus layers are extracted using ultrasonic. The method is economical and simplifies operation, wherein the extraction solution is water, and a large quantity of seed coat mucus layers can be obtained without desalting treatment; and in extraction using an acid-alkali-salt process, residual salt needs to be removed. The time is saved, wherein the extraction time using a vibration process and an alkali salt process is generally 1-2h and desalting is also needed in later period, and the method disclosed by the invention can finish the extraction within 20s. The extraction efficiency is high, wherein a large quantity of residual mucus layers are shown after the seed extracted by the vibration process and acid-alkali-salt process is dyed by ruthenium red; and after the extraction using the method disclosed by the invention, basically no residue of mucus layers exists. The method has the advantages of high efficiency, stability, good reproducibility and the like, can extract the mucus layers to the greatest degree and realize more accurate analysis of the mucus layers, and promotes the study on the formation mechanism of mucus layers; and in the chemical industry, the method can shorten the extraction time and reduce the cost.

Description

technical field [0001] The invention belongs to the field of polysaccharide extraction, in particular to a method for efficiently extracting the inner mucus layer of Arabidopsis thaliana. Background technique [0002] When the embryo of angiosperm develops, the integument covering the embryo will form a seed coat, which isolates various adverse factors from the outside and can regulate the germination of the seed. Some seed plants (such as Cruciferae, Solanaceae, Flax When the seed coat of Plantaceae and Plantagoceae is formed, the outer cells will secrete a large amount of mucus, forming a layer of jelly-like mucus layer around the seed, which plays an important role in promoting the water absorption and diffusion of the seed. Because the mucus layer is rich in pectin, it has become an important raw material for obtaining pectin. The outer cells of Arabidopsis seed coat will accumulate a large amount of mucus between the primary cell wall and the cell membrane during diffe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/00
CPCC08B37/0003C08B37/0057
Inventor 吴蔼民赵先海乔立君陈晓阳
Owner SOUTH CHINA AGRI UNIV
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