Separating and extracting method for thylakoid membranes of C4 plant mesophyll cells and bundle sheath cells

A technology of mesophyll cells and vascular bundle sheaths, applied to plant cells, etc., can solve the problems of increased enzyme usage, poor effect, and long time, and achieve the effects of easy operation, short extraction time, and large processing capacity

Pending Publication Date: 2019-05-07
UNIV OF JINAN
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, enzymatic hydrolysis usually requires a long time of enzymatic hydrolysis to obtain protoplasts, and as the amount of thylakoid membranes to be obtained increases, the amount of enzymes used will also increase, and the cost will also increase
The effect of enzymatic hydrolysis on some mature leaves is not very good

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] a C 4 The method for extracting the mesophyll cell thylakoid membrane and the bundle sheath cell thylakoid membrane of plant maize comprises the following steps:

[0035] (1) Take mature corn leaves (the third leaf and above), wipe off the water after cleaning, remove the veins in the middle of the leaves, and cut the leaves into 0.5-1cm strips;

[0036] (2) Put it in the refrigerator to pre-cool the cup body of the Philips breaker, smashing liquid, swelling liquid, and thylakoid membrane storage liquid; take 50g of cut leaves and put them into the pre-cooled breaker, add 500ml of For mashing liquid, use the smallest file to crush. During this period, samples were taken and observed under a microscope until 85% of the mesophyll cells in the outer layer of the leaves fell off (to ensure the integrity of the vascular bundle sheath cells).

[0037] (3) Place the beaker on ice for pre-cooling, and filter the mixture of corn leaves with a 35-mesh sieve, put the sieve on a ...

Embodiment 2

[0045] The photosystem II light-harvesting antenna complex LHCII is extracted from the thylakoid membrane of maize mesophyll cells, including the following operations:

[0046] Take 5ul of the thylakoid membrane of the mesophyll cell obtained in step (6) of Example 1 and add it to 5ml of 80% acetone, fully extract the chlorophyll, centrifuge at 7000g for 5min, and absorb the supernatant. Measure the absorbance with Japan Shimadzu UV-2600 spectrophotometer, calculate the total concentration of chla+b, use 20mM Tcine-Tirs as the buffer buffer to adjust the final concentration of chla+b in the thylakoid membrane to chl a+b=1mg / mL, 1.0% β-DDM, solubilized in an ice bath at 200 rpm / min for 30 minutes, centrifuged at 20,000 g in a Beckmann high-speed centrifuge JA 25.5 at 4°C for 10 minutes.

[0047] Take the supernatant through the protein purification system, collect and combine multiple collection tubes corresponding to the peak positions of the LHCII samples.

[0048] Manually ...

Embodiment 3

[0052] The pigment-protein complex PSI-LHCI is extracted from the thylakoid membrane of maize vascular sheath cells, including the following operations:

[0053] Take 5ul of the thylakoid membrane of bundle sheath cells obtained in step (7) of Example 1 and add it to 5ml of 80% acetone, fully extract the chlorophyll, centrifuge at 7000g for 5min, and absorb the supernatant. Measure the absorbance with Japan Shimadzu UV-2600 spectrophotometer, calculate the total concentration of chl a+b, use 20mM Tcine-Tirs as buffer buffer to adjust the final concentration of thylakoid membrane chla+b to chla+b=1mg / mL, 0.6% β-DDM, solubilized by stirring in an ice bath at 200rpm / min for 30min, centrifuged at 20000g in a Beckmann high-speed centrifuge JA 25.5 at 4°C for 10min.

[0054] Take the supernatant and collect the collection tube corresponding to PSI-LHCI as the main protein peak through the protein purification instrument.

[0055] Manually pour sucrose density gradient mixture, 0.3-...

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Abstract

The invention belongs to the technical field of biology, and provides a separating and extracting method for thylakoid membranes of C4 plant mesophyll cells and bundle sheath cells. Through the mechanical action of a high-speed blender, thylakoid membranes of C4 plant mesophyll cells and bundle sheath cells can be respectively obtained. The method is simple in technology and easy to operate; the extraction time can be shorter by adopting the technology to extract the thylakoid membranes; compared with an enzyme hydrolysis method, the method is simple in technology, short in extraction time andlarge in processing capacity; and the thylakoid membranes of the C4 plant mesophyll cells and bundle sheath cells can be respectively obtained through the method, so that required proteins can be obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a C 4 A method for separating and extracting thylakoid membranes of mesophyll cells and bundle sheath cells of plants. Background technique [0002] Photosynthesis is the largest energy and material conversion process on the earth, and is the material basis for the survival and development of almost all life. Photosynthesis is divided into two processes. The first process is on the thylakoid membrane. Sunlight excites chlorophyll in the photosynthetic electron transfer system to absorb sunlight, convert light energy into chemical energy, and generate energy molecules ATP and NADPH. This process is called photoreaction. The second process is called the dark reaction. Under the catalysis of enzymes, the energy provided by ATP and NADPH are consumed, and carbon dioxide and water are synthesized into carbohydrates. [0003] Plants can be divided into carbon three plants (C 3 Plants),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
Inventor 林雪乔刘明媚李秀秀秦晓春
Owner UNIV OF JINAN
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