Extraction method of halogeton mesophyll cell vacuoles

An extraction method and technology of halophyte, applied in the direction of plant cells, etc., to achieve the effects of high efficiency, rapid integrity, large quantity and high purity

Active Publication Date: 2016-02-10
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are very few studies on the extraction of halophyte vacuoles, especially about the separation and purification of halophyte vacuoles.

Method used

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  • Extraction method of halogeton mesophyll cell vacuoles
  • Extraction method of halogeton mesophyll cell vacuoles
  • Extraction method of halogeton mesophyll cell vacuoles

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: a kind of method for extracting cell vacuoles of Herba salina leaf, its steps are:

[0026] (1) Leaf sampling: cut and grow 30d seedling leaves, wash 3 times with distilled water, and dry the surface with filter paper;

[0027] (2) Enzymatic hydrolysis and purification of protoplasts: the leaves obtained in step (1) were cut off from the stalks along the base of the leaves, moved to a laboratory table covered with smooth and clean paper, and cut in half along the main veins of the leaves with a blade. Then put in the enzymatic hydrolysis solution, and enzymolyze it for 2.5 hours at a temperature of 25°C in the dark; after the enzymolysis is completed, add an equal volume of washing solution to the enzymatic hydrolysis solution, and place it in dark conditions at a temperature of 6°C and a rotation speed of 30rpm Shake on a shaker for 10 minutes to release the protoplasts; then use a 5ml pipette to transfer all the enzymolysis solution to a test tube, centr...

Embodiment 2

[0033] Embodiment 2: a kind of method for extracting cell vacuoles of Herba salina leaf, its steps are:

[0034] (1) leaf drawing: cut out the leaves of 40d seedlings, wash them with distilled water for 5 times, and dry their surfaces with filter paper;

[0035] (2) Protoplast acquisition and purification. Cut the leaves longitudinally along the main veins with a razor blade, then enzymatically hydrolyze at 22°C for 2.5 hours in the dark, then add an equal volume of washing solution, then shake on a shaker at 6°C and 35rpm for 10 minutes in the dark, Then use a 5ml pipette gun to transfer the enzymolysis solution into a 10ml test tube, then centrifuge at 5°C and 2200rpm for 10min, and use a pipette gun to draw 4ml of the supernatant;

[0036] (3) Acquisition and purification of vacuoles. Add 30ul / ml digitonin to the above supernatant, mix well, let stand at 4°C for 10min, centrifuge at 1200rpm at 4°C for 15min, finally collect 2ml of supernatant, and observe it under an inve...

Embodiment 3

[0037]Embodiment 3: a kind of method for extracting cell vacuoles of Herba salinae blade, its steps are:

[0038] (1) Leaf sampling: cut and grow 25d seedling leaves, rinse 3 times with distilled water, and dry the surface with filter paper;

[0039] (2) Protoplast acquisition and purification. Cut the leaves longitudinally along the main veins with a razor blade, then enzymatically hydrolyze at 25°C for 3.0 hours in the dark, then add an equal volume of washing solution, then shake at 2°C at 40 rpm for 15 minutes in the dark, Then use a 5ml pipette gun to transfer the enzymolysis solution into a 10ml test tube, then centrifuge at 4°C and 2200rpm for 10min, and use a pipette gun to draw 4ml of the supernatant;

[0040] (3) Acquisition and purification of vacuoles. Add 35ul / ml digitonin to the above supernatant, mix well, let stand at 2°C for 15min, centrifuge at 1100rpm at 2°C for 10min, finally collect 2ml of supernatant, and observe it under an inverted microscope. The pr...

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Abstract

The invention belongs to the technical field of extraction of plant cell vacuoles and particularly relates to an extraction method of halogeton mesophyll cell vacuoles. The extraction method of the halogeton mesophyll cell vacuoles is mainly characterized by comprising the following steps: (1) taking mesophyll; (2) enzymolysis and purification of protoplast: cutting the mesophyll obtained in the step (1) from the stalk along the mesophyll base, then putting in an enzymatic hydrolysate, carrying out enzymolysis for 2.5-3.0h at the temperature of 22-25 DEG C under the dark condition, and after the enzymolysis is finished, adding isometric washing liquid into the enzymatic hydrolysate; and (3) obtainment and purification of vacuoles: adding digitonin into the protoplast obtained in the step (2) so as to realize enrichment of pure vacuoles. The digitonin is used for dissociating the plasma membrane, thereby simplifying common complex programs of density gradient centrifugation for extracting the vacuoles. According to the invention, the process of carrying out tender mesophyll protoplast enzymolysis to obtain high-purity vacuoles only takes about 4h. The method realizes simple, efficient and fast extraction of the halogeton mesophyll cell vacuoles. The vacuoles obtained by the method are relatively high in purity, more in quantity, little in impurity contamination and good in integrality.

Description

technical field [0001] The invention belongs to the technical field of extracting plant cell vacuoles, and in particular relates to a method for extracting cell vacuoles from leaves of halophytes halophytes. Background technique [0002] It has become a hot topic in current biological research to study the mechanisms of plant adaptation to stress such as salt tolerance and drought resistance. Halogetonglomeratus grows in the arid region of Northwest my country. It is an annual dilute halophyte of the Chenopodiaceae genus Halogeton. Enrichment, thus forming a special tolerance and adaptation mechanism to salt stress, and its characteristics suitable for artificial cultivation, making halophytic grass a good material for studying the mechanism of salt tolerance and drought resistance in plants. Plant cell vacuole is an organelle with multiple functions, accounting for about 90% of the volume of mature cells. Its main function is to accumulate and store metabolites, regulate os...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
Inventor 王化俊孟亚雄李葆春汪军成姚立蓉马小乐赖勇杨柯司二静任盼荣
Owner GANSU AGRI UNIV
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