Method for rapidly separating glutamine synthetase isoenzymes of crops and rapidly measuring sizes of glutamine synthetase isoenzymes of crops
A glutamine and synthetase technology, applied in biochemical equipment and methods, microbial determination/inspection, measuring devices, etc., can solve problems such as inability to achieve quantitative analysis, unsuitable for soluble protein separation, and expensive equipment
Inactive Publication Date: 2016-11-09
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology
At present, the methods for separating and identifying active proteins mainly include traditional chromatography and centrifugation techniques, which require a large amount of samples and take a long time, and the instruments and equipment are expensive. They are only suitable for qualitative analysis and cannot achieve quantitative analysis.
BNE technology has been successfully applied to the separation and identification of animal mitochondrial membrane proteins, but it is not suitable for the separation of soluble proteins. On the one hand, a large amount of Coomassie brilliant blue in the system leads to protein depolymerization; on the other hand, due to the large amount of RuBP carboxylase in crop leaves Staining interference during electrophoresis, affecting the identification of other proteins
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[0023] The present invention will be further explained below in conjunction with specific embodiments:
[0024] (1) Extraction of total soluble protein from crop tissue
[0025] Weigh 0.5-1 g of fresh leaves, add liquid nitrogen to grind, and add 1-2 mL of extraction buffer (100 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl) 2 and 10mM β-mercaptoethanol, pH 7.6, at 4°C); transfer the tissue homogenate to a 1.5-2mL EP tube, centrifuge at 4°C and 13000g / min for 30min, take the supernatant and place it on ice spare.
[0026] (2) Add the sample, and use the BNE electrophoresis system to separate the total soluble protein of the crop tissue
[0027] First seal the seal with anode buffer and 1% agarose, and then use a gradient meter to make a 4-13% gel gradient. After the separation gel (as shown in Table 1) solidifies, pour a suitable volume of stacking gel (as shown in Table 1). After the gel is prepared, ddH can be used 2 Packed with wet napkins, packed in ziplock bags, and stored at 4°...
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The invention belongs to the technical field of biology, and relates to the technical field of measurement and identification of protein polymer molecular sizes, in particular to a method for rapidly separating glutamine synthetase isoenzymes of crops and rapidly measuring the sizes of the glutamine synthetase isoenzymes of the crops. The method includes the following steps that crop tissue soluble total protein is extracted; the crop tissue soluble total protein is separated through an improved BNE electrophoresis system; the activity of the glutamine synthetase isoenzymes in gel is measured, and a gel image are scanned; after the gel is subjected to Coomassie brilliant blue dyeing, an image is scanned, the two images are combined, and the sizes of the glutamine synthetase isoenzymes are analyzed through software; according to the sizes and sub-unit properties of the glutamine synthetase isoenzymes, the polymerization status of the glutamine synthetase isoenzymes is analyzed and calculated. The sizes and polymerization status grade expression regulation condition of GS isoenzyme holoenzymes of the crops can be rapidly known, and a molecular foundation is provided for breeding nitrogen high-efficiency varieties.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to the technical field of protein polymer molecular size determination and identification, in particular to a method for rapidly separating and determining the size of crop glutamylamine synthase isozymes. Background technique [0002] Nitrogen is the main element that affects crop yield and quality. Whether it comes from nitrate, ammonia ions, microbial fixation of nitrogen or ammonia released during plant metabolism, it must be catalyzed by glutamine synthase (GS) to be assimilated into amino acids. . There are two types of GS in higher plants, one is cytosolic GS, namely GS1, which is mainly involved in the transport of stored nitrogen sources during seed germination and the transfer and reuse of nitrogen sources during leaf senescence; the other is plastid-type GS, namely GS2 is mainly involved in the assimilation process of ammonia (primary nitrogen) produced by photorespiration and...
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IPC IPC(8): C12Q1/25
CPCC12Q1/25G01N2333/9015
Inventor 王小纯韦一昊张浩然马新明
Owner HENAN AGRICULTURAL UNIVERSITY