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A method for extracting sedum plant cell vacuoles

A technology of plant cells and Crassulaceae, applied in plant cells, etc., can solve the problems of low vacuole efficiency, inability to obtain complete and pure vacuoles, etc., and achieve the effect of good integrity, large number of vacuoles, and high purity

Active Publication Date: 2015-09-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the process of vacuole purification in this method, the crude extraction vacuole is first resuspended in a polysucrose solution with a higher concentration, and then a layer of polysucrose solution with a lower concentration is added to the upper layer of the resuspension for density gradient centrifugation to extract The efficiency of the vacuole is low, and even the complete and pure vacuole cannot be obtained

Method used

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  • A method for extracting sedum plant cell vacuoles
  • A method for extracting sedum plant cell vacuoles
  • A method for extracting sedum plant cell vacuoles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1 solution preparation

[0051] (1) Cell lysate: 15g cellulase, 4g isolated enzyme, 72.88g mannitol, 1.49g potassium chloride, 4.264g morpholine ethanesulfonic acid monohydrate, 1.11g calcium chloride, 0.39065g β-mercaptoethanol, dissolved Dilute to 1L in ultrapure water;

[0052] (2) Protoplast protection solution: 0.1543g sodium chloride, 13.875g calcium chloride, 0.373g potassium chloride, 0.426g morpholinoethanesulfonic acid monohydrate, dissolved in ultrapure water and dilute to 1L;

[0053] (3) Protoplast lysate: 0.083g mannitol, 0.173g ethylene glycol bis-2-aminoethyl ether (EGTA), 0.559g 3-[(3-cholesterolaminopropyl)dimethylamino]-1-propane Sulfonic acid (CHAPS), 3.876g morpholineethanesulfonic acid monohydrate, 12.614g calcium chloride, 0.339g potassium chloride, dissolved in 950mL ultrapure water, adjusted to pH 8.0 with 2M Tris solution, and then dilute to 1L;

[0054] (4) Vacuole protection solution: 4.264g morpholineethanesulfonic acid monohydrate, 145.7...

Embodiment 2

[0077] Utilize the same method and reagents as in Example 1 to carry out vacuole extraction, but add 7% polysucrose solution to the bottom of the centrifuge tube when purifying the vacuole, carry out density gradient centrifugation, and take the transition layer between the resuspension and 7% polysucrose solution (not A clear pink layer is formed) liquid microscope examination, the results of the microscope examination are shown in Image 6 .

[0078] Depend on Image 6 It can be seen that there are only a few red vacuoles with transparent edges, most of which are protoplasts or vacuoles with ruptured protoplast membranes adhered to the surface, and the degree of vacuole purification is not high.

Embodiment 3

[0080] Utilize the same method and reagents as in Example 1 to carry out vacuole extraction, but add 4% polysucrose solution to the bottom of the centrifuge tube when purifying the vacuole, carry out density gradient centrifugation, and take the transition layer between the resuspension and 4% polysucrose solution (the layer is very inconspicuous) liquid microscopic examination, the results of microscopic examination are shown in Figure 7 .

[0081] Figure 7 In the medium, only protoplasts or vacuoles with ruptured protoplast membranes adhered to the surface were seen, and no red vacuoles with transparent edges were seen.

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Abstract

The invention discloses a method for extraction of Crassulaceae plant cell vacuoles. The method includes protoplast extraction, vacuole coarse extraction, and vacuole purification. The vacuole purification includes: resuspending the vacuoles obtained by coarse extraction in a density gradient medium solution with a mass / volume concentration of 0-5%, adding a density gradient medium solution with a mass / volume concentration of 7-10% into the lower layer of the resuspension solution, and carrying out density gradient centrifugation, thus obtaining purified vacuoles. In the invention, during vacuole purification, firstly the density gradient medium solution with a small concentration is utilized to resuspend the coarsely extracted vacuoles in a centrifuge tube, then the density gradient medium solution with a large concentration is slowly added into the centrifuge tube bottom along the centrifuge tube wall, the solution in the centrifuge tube falls into two layers at the moment, and then density gradient centrifugation is performed. The obtained vacuoles have the advantages of high membrane integrity, large quantity, and high purity.

Description

technical field [0001] The invention belongs to the technical field of plant cell vacuoles extraction, and in particular relates to a method for extracting sedum plant cell vacuoles. Background technique [0002] Sedum sedum is one of the few cadmium hyperaccumulator plants found in China. It belongs to Crassulaceae. It also has strong tolerance and accumulation ability to zinc and lead. It has great application prospects in the field of phytoremediation of heavy metal polluted soil in my country. The research on the superaccumulation characteristics and physiological mechanism of Sedum sedum can not only promote the direct application of this plant in the field of remediation of polluted environment, but also help to prove the phenomenon of superaccumulation of heavy metals in plants, so as to promote the improvement of phytoremediation technology in polluted soil And optimization provides a solid theoretical basis. In view of this, Sedum asiatica is a polymetallic accumul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
Inventor 卢玲丽廖星程田生科谢若瀚韦燕燕
Owner ZHEJIANG UNIV
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