An electrophoresis method for removing the interference of rubisco enzyme from watermelon leaves and separating the remaining low-abundance proteins

A protein and watermelon technology, applied in the biological field, can solve the problems of cumbersome and complicated operations, need to be further investigated and verified, and not identical.

Inactive Publication Date: 2015-08-26
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is tedious and complicated to operate, and its removal operation needs to be carried out at high temperature (37°C or 42°C), which may lead to the degradation and loss of other proteins, especially some regulatory factors and signaling factors (Krishnan and Natarajan, 2009; Li Hongbing and Kang Zhensheng , 2011)
3) High-concentration dithiothreitol (DTT) precipitation method (Cho et al., 2008), its principle and effectiveness need further investigation and verification
However, due to the molecular weight and isoelectric point of proteins associated with the use of PEG to precipitate proteins, there are certain differences in the molecular weight and isoelectric point of Rubisco enzymes of different plant leaves (Chai Changxing et al., 1985). Therefore, 15% or 16% PEG is used to remove rice Leaf Rubisco method does not work on watermelon leaves
In addition, monocot rice leaves contain a lot of cellulose and lignin, each component and content are different from the protein composition of watermelon leaves, and the method of extracting the remaining protein by phenol extraction or acetone precipitation is not the same The best way to boost protein in watermelon leaves

Method used

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  • An electrophoresis method for removing the interference of rubisco enzyme from watermelon leaves and separating the remaining low-abundance proteins
  • An electrophoresis method for removing the interference of rubisco enzyme from watermelon leaves and separating the remaining low-abundance proteins

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Embodiment 1

[0028] Example 1 Screening of the best PEG concentration of Rubisco in precipitation of watermelon leaves

[0029] A. Take 2g of mature leaves of watermelon (Citrullus Ianatus Mansfeld) seedlings, place them in liquid nitrogen and quickly grind them to a fine powder (particle size is about 0.01mm). Use a small spatula to quickly transfer the powder to a 10ml centrifuge tube, and add 7ml Cold homogenization buffer, then shake and mix on a vortexer, and incubate horizontally on ice for 10 minutes.

[0030] B. Centrifuge at 1500g for 3 minutes at 4°C, discard the precipitate and save the supernatant; then centrifuge at 1,500g at 4°C for 20 minutes, discard the precipitate and save the supernatant.

[0031] C. Add different concentrations of PEG-4000 to the supernatant, incubate horizontally on ice for 20 minutes, centrifuge at 1,500g at 4°C for 25 minutes, discard the precipitate, and save the supernatant.

[0032] D. Quantify the protein in the supernatant obtained in step C to 2ug / ul b...

Embodiment 2

[0035] 1) Preparation of remaining protein samples after precipitation of Rubisco of watermelon leaves

[0036] A. Repeat step AB of Example 1 to obtain the whole protein supernatant. Add 60% PEG-4000 to the supernatant to make the final concentration of PEG-4000 in the solution reach 18%. Vibrate and mix well on a vortex. Incubate on ice for 20 minutes, centrifuge at 1,500g at 4°C for 25 minutes, discard the pellet, and save the supernatant.

[0037] B. Add 8 volumes of 10% trichloroacetic acid-acetone solution (w / v, containing 0.07% β-mercaptoethanol) to the supernatant, and place it at -20°C overnight. Centrifuge at 2,500g for 25 minutes at 4°C, discard the supernatant, and save the precipitate.

[0038] C. Add 8ml of acetone (containing 0.07% β-mercaptoethanol) to the precipitate to clean the precipitate, place it at -20°C for 1 hour, centrifuge under the same conditions as in step B, discard the supernatant, and save the precipitate.

[0039] D. Repeat step C 3-4 times until the...

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Abstract

The invention belongs to the technical field of biology, and discloses an electrophoresis method for removing Rubisco from plant leaves and enriching and separating residual proteins. The method comprises the following steps of: removing Rubisco from watermelon leaves by using 18 percent PEG-4000; leaching residual proteins through a TCA-acetone precipitation method; and performing two-dimensional electrophoresis to separate residual proteins from watermelon leaves. Compared with a holoprotein two-dimensional electrophoretogram, the method has the advantages that: after Rubisco in watermelon leaves is precipitated by adopting the 18 percent PEG, most large and small subunits of Rubisco disappear, the expression levels of most residual proteins are raised, the quantity of proteins obtained by separating is increased by 48.4 percent, and a large quantity of new protein spots (65+ / -15) appear in a region of which the molecular weight is smaller than 25.0kDa. As proved by repeated experiments, the electrophoresis method has the advantages of high repeatability, clear spectra and reliable result.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to an electrophoresis method for removing Rubisco enzyme interference from plant leaves, enriching and separating remaining low-abundance proteins, and more particularly relates to an electrophoresis method for removing Rubisco enzyme interference from watermelon leaves, enriching and separating low-abundance proteins. Electrophoresis method for abundant proteins. Background technique [0002] Watermelon is one of the important economic crops in my country, and it is also an important fruit-type vegetable in the world. Studying its stress resistance mechanism and nutritional quality functional factors is especially important for the cultivation and production of watermelon. Watermelon leaf proteomics research can provide important functional information for watermelon resistance cultivation and nutritional quality analysis. Protein two-dimensional electrophoresis is the most commonly used tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C07K1/28C07K1/26
Inventor 郭世荣阳燕娟孙锦严蓓何立中李斌
Owner NANJING AGRICULTURAL UNIVERSITY
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