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Method for extracting protein from cotton leaves

An extraction method and protein technology, applied in the field of biochemistry, can solve problems affecting protein purity, affecting the development of cotton industry, affecting the research of cotton protein field, etc., and achieve the effect of good repeatability, stable protein and high protein purity

Inactive Publication Date: 2014-03-26
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cotton contains a large number of secondary metabolites such as pigments, phenols, and quinones, which seriously affect the purity of the extracted protein, thereby affecting the research in the field of cotton protein and the development of the cotton industry.

Method used

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  • Method for extracting protein from cotton leaves
  • Method for extracting protein from cotton leaves
  • Method for extracting protein from cotton leaves

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Cotton leaf obtains

[0027] Select cotton seeds with full grains and clean fluff, put them into a triangular flask, soak the seeds for 24 hours, and transfer them to a germination box with two layers of filter paper after the seeds are white, and keep the germination box moist until two cotyledons grow. Finally, the vigorous and uniform cotton seedlings were selected and transferred to 1 / 2 Hoagland culture medium, and hydrocultured in an artificial climate chamber. The culture conditions are as follows: temperature is 28 / 25C (day / night), light intensity is above 12000 lx, light is 350-400, light is 10 hours a day, and relative humidity is 60-80%. Change the culture medium every 4 days, and pay attention to cleaning the moss on the roots and pots, etc. After cultivating for half a month, take the leaves, wash them with pure water and deionized water in turn, dry the water with filter paper, and put them in liquid nitrogen After quick-freezing, store in a -...

Embodiment 2

[0029] Example 2 Cotton leaf protein extraction

[0030] Burn the cleaned mortar with an alcohol lamp for three minutes, put it in a -20°C refrigerator for pre-cooling, add 1-2g of materials, an appropriate amount of PVPP and a little quartz sand, and grind thoroughly with liquid nitrogen until the powder turns white. Transfer to several 2mL centrifuge tubes (about 0.4g per tube), add 1.8mL pre-cooled trichloroacetic acid extract to each tube, mix well, put in -20°C refrigerator for more than two hours, and leave for about half an hour Shake once. Centrifuge at 13000r / min for 15min at 4°C, discard the supernatant, and collect the precipitate. After crushing the precipitate with a pipette tip, add 1.8mL of -20°C pre-cooled acetone (containing 0.07%-0.1%DTT, 1mmol / LPMSF), mix well and let stand at -20°C for 2-4h. Centrifuge as above, discard Take the supernatant, and take the precipitate, and repeat the washing 3 times. Finally, take the precipitate, if you find that there is...

Embodiment 3

[0039] Extracted protein leaf protein purity detection of embodiment 3

[0040] Detection method:

[0041] 1. Protein Quantification

[0042] Use 1% BAS (Bovine Serum Albumin) as standard protein, 0.01% Coomassie Brilliant Blue G250, 5% ethanol (concentration: 95%), 10% phosphoric acid staining, measure the OD value at a wavelength of 595nm on a UV spectrophotometer and draw standard curve line. Calculate the protein concentration of the sample according to the standard curve.

[0043] 2. Hydration

[0044]After thawing the protein sample placed in an ultra-low temperature refrigerator at room temperature, add hydration buffer (7mol / L urea, 2mol / L thiourea, 4% CHAPS, 80mmol / LDTT, 1mmol / LPMSF, 0.3% carrier to the centrifuge tube Ampholyte (PH4-7), mass fraction 0.002% bromophenol blue). In this experiment, IPG strips with pH 3-10 were used to separate the total protein of cotton leaves, and it was found that the protein spots were mainly concentrated between pH 5-8 (resu...

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Abstract

The invention provides a method for extracting protein from cotton leaves. The method comprises the following steps of 1) grinding the cotton leaves; 2) transferring the ground leaves powder to a centrifugal pipe, adding trichloroacetic acid extraction liquid, uniformly mixing, and storing the mixture in a refrigerator with the temperature of -20 DEG C for more than 2h; 3) centrifuging in the centrifugal pipe, removing the supernatant, retaining the precipitate, adding acetone solution pre-cooled at the temperature of -20DEG C into the precipitate, uniformly mixing, and standing for 2h to 4h at the temperature of -20DEG C; 4) centrifuging in the centrifugal pipe, removing the supernatant, retaining the precipitate, washing the precipitate, and retaining the precipitate; 5) vacuumizing the precipitate, and volatizing acetone to obtain dry protein powder; 6) adding protein lysate into the dry protein powder, disintegrating for 1h in the water bath with temperature being 35DEG C, centrifuging, and taking the supernatant which is the cotton leave protein dissolved liquid. By adopting the method for extracting the protein from the cotton leaves, the extracted protein is stable, the repeatability is good, the purity of the protein is high, and the electrophoretogram show that no grain phenomena such as the cross stripes and streaking appear.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for extracting protein from cotton leaves. Background technique [0002] Cotton is the most important natural fiber crop in the world, and it is also an important oil crop; at the same time, cotton is also an important economic crop worldwide, occupying an important position in the national economy. Cotton can be made into various specifications of textiles, which are comfortable to wear, durable, washable and ironed at high temperatures. Therefore, cotton has great application value and development potential. [0003] Cotton is an important economic and oil crop. After a large number of studies on molecular markers and QTL, it is very urgent and more feasible to conduct proteome research on it. However, cotton contains a large number of secondary metabolites such as pigments, phenols, and quinones, which seriously affect the purity of the extracted protein, thereby affecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K1/30
CPCC07K1/30
Inventor 曲延英陈全家郭忠军孙国清
Owner XINJIANG AGRI UNIV
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