Preparation method of wheat complete chloroplasts

A chloroplast and wheat technology, applied in the field of preparation of complete wheat chloroplasts, can solve the problems of long time consumption, loss of chloroplast DNA, inability to obtain sufficient matrix protein, etc., and achieve the effect of short preparation time and simple procedure

Inactive Publication Date: 2016-08-31
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The traditional separation and purification of chloroplasts often use sucrose density gradient centrifugation and differential centrifugation, which not only takes a long time, but the density gradient centrifugation alone takes more than 5 hours, and this method cannot distinguish between intact and damaged chloroplasts ( figure 1 B), inability to obtain sufficient matrix protein may lead to loss of chloroplast DNA, seriously affecting follow-up research

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  • Preparation method of wheat complete chloroplasts

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Experimental program
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Effect test

Embodiment 1

[0020] 1. Prepare Percoll gradient:

[0021] (1) Weigh 10 mg of erythorbic acid and glutathione (stored at 4°C) each and put into a 50 mL round-bottomed translucent polyethylene centrifuge tube;

[0022] (2) Add 15mL 2×GB (grinding buffer pH7.3, containing 0.1M K-Hepes, 0.66M sorbitol, 2mM MgCl 2 , 2mM MnCl 2 , 4mM Na 2 EDTA and 0.2% BSA, stored at 4°C) for dissolution;

[0023] (3) Add 15 mL Percoll (stored at 4°C) and mix well;

[0024] (4) Centrifuge at 4°C and 37,000g for 30 minutes (note: brake on). After the centrifugation is completed, carefully remove the centrifuge tube and place it on ice for later use.

[0025] 2. Chloroplast preparation (can be performed synchronously during centrifugation in step 1, and all operations are performed at low temperature):

[0026] (1) Vessel preparation: Take two 250mL Erlenmeyer flasks labeled GB and IB respectively, four 50mL round-bottomed centrifuge tubes, one beaker, one plastic funnel, and one pair of scissors and place th...

Embodiment 2

[0039] 1. Prepare Percoll gradient:

[0040] (1) Weigh 10 mg of erythorbic acid and glutathione (stored at 4°C) each and put into a 50 mL round-bottomed translucent polyethylene centrifuge tube;

[0041] (2) Add 15mL 2×GB (grinding buffer pH7.3, containing 0.1M K-Hepes, 0.66M sorbitol, 2mM MgCl 2 , 2mM MnCl 2 , 4mM Na 2 EDTA and 0.2% BSA, stored at 4°C) for dissolution;

[0042] (3) Add 15 mL Percoll (stored at 4°C) and mix well;

[0043] (4) Centrifuge at 4°C and 37,000g for 30 minutes (note: brake on). After the centrifugation is completed, carefully remove the centrifuge tube and place it on ice for later use.

[0044] 2. Chloroplast preparation (can be performed synchronously during centrifugation in step 1, and all operations are performed at low temperature):

[0045] (1) Vessel preparation: Take two 250mL Erlenmeyer flasks labeled GB and IB respectively, four 50mL round-bottomed centrifuge tubes, one beaker, one plastic funnel, and one pair of scissors and place them...

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Abstract

The invention belongs to the technical field of biotechnology, and relates to the technical field of plant complete cell organ separation, in particular to a simple and rapid preparation method of wheat complete chloroplasts. The method comprises the following steps of 1, preparing a Percoll gradient; 2, preprocessing a wheat sample; 3, performing density gradient centrifuging on the wheat sample; 4, completing extraction of the chloroplasts. The Percoll is adopted as a medium for gradient centrifuging, wheat complete and damaged chloroplasts can be distinguished, the preparation time is short, the procedure is simple and convenient, the sufficient and complete chloroplasts are obtained, quantitative determination is performed, and then the chloroplasts can be precisely applied to extraction of chloroplast function protein, membrane protein, matrix protein and chloroplast DNA and various follow-up researches.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to the technical field of separation and purification of complete plant organelles, and in particular relates to a simple and quick preparation method of complete wheat chloroplasts. Background technique [0002] Chloroplast is the most important organelle of plant cells. It is the place where plants carry out photosynthesis and nitrogen assimilation. It is the original source of all organic matter in nature. It is closely related to plant growth and crop yield. Obtaining complete chloroplasts is very important for studying chloroplast functions and chloroplasts. A necessary condition for soluble protein and membrane protein, chloroplast gene, etc. The traditional separation and purification of chloroplasts often use sucrose density gradient centrifugation and differential centrifugation, which not only takes a long time, but the density gradient centrifugation alone takes more than 5...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04C12N2509/00
Inventor 王小纯张浩然韦一昊熊淑萍马新明
Owner HENAN AGRICULTURAL UNIVERSITY
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