Quick extraction method of high-activity mitochondria in plant organs and special extracting solution thereof

A plant organ and extraction method technology, applied to plant cells and other directions, can solve the problems of research lag and complicated extraction methods, and achieve the effects of long storage time, wide applicability and simple operation.

Active Publication Date: 2018-08-07
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research systems for extracting highly active mitochondria for respiration, energy metabolism and superoxide levels are mostly reported in animal studies, and the extraction technology in cardiomyocytes and skeletal muscle cells is more mature; however, in plants, extracting mitochondria The research objects mainly focus on the model plants Arabidopsis thaliana, rice and their single-cell pollen tubes, etc., and the extraction method is too cumbersome
Most of the plant species in the current research are non-model pl

Method used

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  • Quick extraction method of high-activity mitochondria in plant organs and special extracting solution thereof
  • Quick extraction method of high-activity mitochondria in plant organs and special extracting solution thereof
  • Quick extraction method of high-activity mitochondria in plant organs and special extracting solution thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1, the extraction and detection of lotus receptacle mitochondria

[0054] 1. Extraction of Mitochondria from Lotus Receptacle

[0055] 1. Take the fresh lotus receptacle and cut it into small pieces with a blade (the volume is about 1mm 3 ) to get the lotus receptacle fragments.

[0056] 2. Take 1-2g lotus receptacle fragments, add 2-4mL 4°C pre-cooled extract and appropriate amount of quartz sand, then put it in a mortar and grind until homogenized, filter with nylon mesh (50μm), and collect the filtrate.

[0057] 3. Take the filtrate collected in step 2, centrifuge at 2000 g for 10 min at 4°C, and collect the supernatant.

[0058] 4. Take the supernatant collected in step 3, centrifuge at 12000g for 20min at 4°C, and collect the precipitate. The precipitate is the extracted lotus receptacle mitochondria.

[0059] Add 300 μL of 4°C pre-cooled mitochondrial respiration buffer to the precipitate collected in step 4, and resuspend to obtain the mitochondria re...

Embodiment 2

[0065] Example 2, Extraction and Detection of Magnolia Style Mitochondria

[0066] 1. Take fresh magnolia stalks and cut them into small pieces (about 1mm in volume) with a blade 3 ), to obtain Magnolia style fragments.

[0067] 2. Take 1-2g of Magnolia style fragments, add 2-4mL 4 ℃ pre-cooled extract and appropriate amount of quartz sand, then put it in a mortar and grind until homogenized, filter with nylon mesh (specification: 50μm), and collect the filtrate .

[0068] 3. Take the filtrate collected in step 2, centrifuge at 2000 g for 10 min at 4°C, and collect the supernatant.

[0069] 4. Take the supernatant collected in step 3, centrifuge at 12000g for 20min at 4°C, and collect the precipitate.

[0070] 5. Take the precipitate collected in step 4, add 1 mL of 4°C pre-cooled resuspension buffer, and resuspend to obtain a resuspension.

[0071] 6. Take the resuspension obtained in step 5, centrifuge at 1500g at 4°C for 5min, and collect the supernatant.

[0072] 7. T...

Embodiment 3

[0079] Example 3, Extraction and Detection of Lotus Tepal Mitochondria

[0080] 1. Extraction of mitochondria from lotus flower tepals

[0081] 1. Take fresh lotus tepals and cut them into small pieces with a blade (the volume is about 1mm 3 ), to obtain fragments of lotus tepals.

[0082] 2. Take 1-2g lotus perianth fragments, add 2-4mL 4°C pre-cooled extract and appropriate amount of quartz sand, then put it in a mortar and grind until homogenized, filter with nylon mesh (specification: 50μm), collect filtrate.

[0083] 3. Take the filtrate collected in step 2, centrifuge at 2000 g for 10 min at 4°C, and collect the supernatant.

[0084] 4. Take the supernatant collected in step 3, centrifuge at 12000g for 20min at 4°C, and collect the precipitate. The precipitate is the extracted lotus tepal mitochondria.

[0085] Add 300 μL of 4°C pre-cooled mitochondrial respiration buffer to the precipitate collected in step 4, and resuspend to obtain a mitochondrial resuspension of...

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Abstract

The invention discloses a quick extraction method of high-activity mitochondria in plant organs and special extracting solution thereof. The quick extraction method comprises the following steps: mixing the plant organs, the extracting solution and grinding medium, homogenizing, collecting filtrate, carrying out differential centrifugal separation on the mitochondria in the plant organs, and thenadopting mitochondrial respiratory buffer solution for storage. The extracting solution is prepared from sucrose, sodium pyrophosphate, EDTA (Ethylene Diamine Tetraacetic Acid), potassium dihydrogen phosphate, BSA (Bovine Serum Albumin), cysteine, ascorbic acid and pvp-40. Proved by the experiment, the quick extraction method disclosed by the invention has the following advantages that the efficiency is high, i.e., the activity of the mitochondria is high; the applicability is wide, so that the quick extraction method can be applicable to extraction of the mitochondria of flower organs of multiple angiosperms; the storage time is long, and the activity can be maintained for 5 hours on an ice box, so that a great number of studies on the energy metabolism and the respiration of the mitochondria can be met; the operation is simple and the consumed time is short; the method provided by the invention lays a solid foundation for study on the respiratory chain and the energy metabolism of the mitochondria and has important practical application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a rapid extraction method of highly active mitochondria in plant organs and a special extraction solution thereof. Background technique [0002] As one of the important characteristics of angiosperms, flowers are composed of sepals, petals, stamens (stamen group) and pistils (gynosperm group), which are closely related to the reproductive process of plants and are important for ensuring the pollination, fertilization and smooth fruiting of angiosperms. organ. Therefore, floral organs have attracted much attention as the key object of plant reproductive biology research. Moreover, as the flower organ is an important place for plant reproduction and reproduction, its life activities are vigorous, and mitochondria, as an important organelle of energy metabolism, can perform physiological activities such as oxidative phosphorylation and electron transfer in response to diffe...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 王若涵张出兰刘丽娅李静王众司
Owner BEIJING FORESTRY UNIVERSITY
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