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Proline Subcellular Isolation Method

A proline subcellular and separation method technology, applied in the field of proline subcellular separation, can solve the problems of low stability of dithiothreitol and the like

Active Publication Date: 2020-08-04
GUILIN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Tris-HCl centrifugation is mainly used for plant subcellular separation. The effective pH range of Tris-HCl buffer solution is between 7.0-9.2. The specific function of Tris is as an anti-acid component to prevent the pH of the solution from dropping significantly; Salt maintains a certain osmotic pressure to avoid organelle breakage; dithiothreitol has strong reducing properties in alkaline environment, which can prevent the formation of disulfide bonds in protein molecules or between molecules; at the same time, dithiothreitol has low stability, Need to use in low temperature environment

Method used

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  • Proline Subcellular Isolation Method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A method for subcellular separation of proline, specifically carried out according to the following steps:

[0024] Step 1, take 0.5g rice seedling sample and grind it into a homogenate in 6ml of 3% sulfosalicylic acid in an ice bath;

[0025] Step 2, centrifuge the grinding liquid obtained in step 1 at 3000r / min at 4°C for 5min at low temperature; save the lower sediment a for later use, centrifuge the supernatant a at 10000r / min for 30min; supernatant b is the cell solution group Points were used to analyze proline; precipitation b was reserved for future use.

[0026] Step 3, dissolve and mix the precipitates a and b in step 1 with 5ml Tris-HCl solution, the Tris-HCl solution is 50mM Tris-HCl pH 7.5, 0.25mM sucrose, 1mM dithiothreitol;

[0027] Step 4, take 600 g of the homogenate obtained in step 3 and centrifuge at a low temperature of 4°C for 1 min at a speed of 2500 r / min, the sediment c is the cell wall part; the supernatant c is the organelle part;

[0028] S...

Embodiment 2

[0033] A method for subcellular separation of proline, specifically carried out according to the following steps:

[0034] Step 1, take 0.4g rice seedling sample and grind it into a homogenate in 5ml of 3% sulfosalicylic acid in an ice bath;

[0035] Step 2, centrifuge the grinding liquid obtained in step 1 at 3000r / min at 4°C for 5min at low temperature; save the lower sediment a for later use, centrifuge the supernatant a at 10000r / min for 30min; supernatant b is the cell solution group Points were used to analyze proline; precipitation b was reserved for future use.

[0036] Step 3, dissolve the precipitates a and b in step 1 with 4ml Tris-HCl solution and mix evenly, the Tris-HCl solution is 50mM Tris-HCl pH 7.5, 0.25mM sucrose, 1mM dithiothreitol;

[0037] Step 4, take 600 g of the homogenate obtained in step 3 and centrifuge at a low temperature of 4°C for 1 min at a speed of 2500 r / min, the sediment c is the cell wall part; the supernatant c is the organelle part;

[...

Embodiment 3

[0043] A method for subcellular separation of proline, specifically carried out according to the following steps:

[0044] Step 1, take 0.6g rice seedling sample and grind it into a homogenate in 5ml of 3% sulfosalicylic acid in an ice bath;

[0045] Step 2, centrifuge the grinding liquid obtained in step 1 at 3000r / min at 4°C for 5min at low temperature; save the lower sediment a for later use, centrifuge the supernatant a at 10000r / min for 30min; supernatant b is the cell solution group Points were used to analyze proline; precipitation b was reserved for future use.

[0046] Step 3, dissolve the precipitates a and b in step 1 with 6ml Tris-HCl solution and mix well, the Tris-HCl solution is 50mM Tris-HCl pH 7.5, 0.25mM sucrose, 1mM dithiothreitol;

[0047] Step 4, take 600 g of the homogenate obtained in step 3 and centrifuge at a low temperature of 4°C for 1 min at a speed of 2500 r / min, the sediment c is the cell wall part; the supernatant c is the organelle part;

[0048...

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Abstract

The invention discloses a method for separating and extracting proline in sub-cells of plants. A rice seedling sample is taken and grinded into homogenate in sulfosalicylic acid through ice bath; grinding liquid is centrifuged for 5 minutes; lower-layer precipitate a is put for standby application; supernate a is centrifuged for 30 minutes to obtain supernate b and precipitate b; dissolution and mixing are performed by using a Tris-HCl solution; 600 g of homogenate is centrifuged at the temperature of 4 DEG C, and precipitate c is a cell wall part; supernate c is an organelle part; the supernate c and the precipitate c are respectively centrifuged for 30 minutes, and precipitate d and precipitate e are retained respectively; 3% sulfosalicylic acid is added respectively for dissolution, centrifugation continues, and then precipitate f and precipitate g at the lower portion are retained respectively; 3% sulfosalicylic acid is added respectively for dissolution, ultrasonic treatment is performed, boiling water bath is performed for 30 minutes, centrifugation is performed at the temperature of 4 DEG C for 10 minutes, and obtained supernate d and supernate e are an organelle component and a cell wall component. The proline content in a subcellular structure can be conveniently and directly determined.

Description

technical field [0001] The invention belongs to the technical field of plant cell separation and relates to a proline subcellular separation method. Background technique [0002] Since Kemble and MacPherson first discovered in 1954 that the detached leaves of ryegrass would produce and accumulate a large amount of free proline when wilting, the research on plant proline began to attract attention. Proline is one of the components of plant protein, and can widely exist in plants in a free state. [0003] Under natural conditions, plants are often subjected to osmotic stress caused by adversity environments such as drought, high salinity, and high temperature. Many plants accumulate proline in response to osmotic stress. Proline is a compatible osmotic adjustment substance. As an ideal osmotic adjustment substance, proline has the characteristics of small molecular weight and easy solubility in water. As a cell structure protector and free radical scavenger, proline also pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D207/16G01N21/78
CPCC07D207/16G01N21/78
Inventor 于晓章卢明锐
Owner GUILIN UNIVERSITY OF TECHNOLOGY
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