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Construction method of soybean seed protein expression profile and application of soybean seed protein expression profile to analysis of biological metabolism ways

A technology of soybean protein and construction method, applied in the field of protein expression profile construction, can solve the problem that there is no research on the identification of the whole protein of soybean dried seeds.

Pending Publication Date: 2019-04-09
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the field of soybean seed proteomics, Mooney et al. have studied the two-dimensional electrophoresis pattern of soybean mature dry seeds, but only identified part of the protein, and there is no research on the identification of soybean dry seed full protein

Method used

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  • Construction method of soybean seed protein expression profile and application of soybean seed protein expression profile to analysis of biological metabolism ways
  • Construction method of soybean seed protein expression profile and application of soybean seed protein expression profile to analysis of biological metabolism ways
  • Construction method of soybean seed protein expression profile and application of soybean seed protein expression profile to analysis of biological metabolism ways

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Embodiment 1

[0074] 1 Materials and methods

[0075] 1.1 Experimental materials

[0076] The material used in this study is Zhongpin 661, which was planted in Shunyi District, Beijing, and the seeds were harvested in October 2016 for experimentation.

[0077] 1.2 Method

[0078] 1.2.1 Extraction of total protein from Zhongpin 661 soybean seeds

[0079] After the mature seeds of Zhongpin 661 were naturally air-dried (the water content of the seeds was about 12%), the cotyledons on the back of the hilum of soybean were ground with a carving knife to obtain soybean powder, and 5 mg of soybean powder was weighed, and 500 μl of extraction buffer (0.05 mol / L Tris (Tris, 2% sodium dodecyl sulfate SDS, 5mol / L urea, 0.1% Bromophenol blue, pH8.0), add 10 μl mercaptoethanol, shake for 5 minutes on a shaker , centrifuged at 12000r / min for 10 minutes, and the supernatant was extracted as the total protein.

[0080] 1.2.2 SDS-PAGE electrophoresis

[0081] A discontinuous vertical electrophoresis a...

Embodiment 2

[0113] Thylakoid membrane protein was extracted from the young leaves of soybean seed coat color near-isogenic lines 3388 (yellow) and 3385 (green) (add about 5 ml of pre-cooled extraction buffer per 1 g of soybean leaves, and the extraction buffer was 0.4 mol / L sucrose, 0.01mol / L NaCl, 0.002mol / L MgCl 2 ·6H 2 O, 0.05mol / L Hepes, pH=7.8) and liquid enzyme digestion, and then perform segmental mass spectrometry analysis. According to the molecular weight, the protein is divided into 3 segments: the molecular weight is less than 25KDa, and the molecular weight is between 25KDa and 45KDa (see image 3 ), the molecular weight is greater than 45KDa, and the proteins whose molecular weight is not in the selected area are excluded. A total of 2170 proteins were detected in the green seed coat 3385, and a total of 1730 proteins were detected in the yellow seed coat 3388. Among them, the proteins specifically expressed in the green seed coat 1140 kinds of proteins, 700 kinds of prot...

Embodiment 3

[0120] Soybean material is JACK, yellow seed coat 3388 and green seed coat 3385. Take 1g of soybean leaves respectively, add about 5ml of pre-cooled extraction buffer (0.4mol / L sucrose, 0.01mol / L NaCl, 0.002mol / L MgCl 2 ·6H 2 O, 0.05mol / LHepes, pH=7.8), ground in an ice bath until homogenized, filtered with 4 layers of lens paper, centrifuged at 5000g at 4°C for 10min, and the thylakoid membrane was washed with buffer (0.33M Sorbitol, 0.05M Bis-Tris, pH = 7.0) was washed several times until no impurities, and then suspended with an appropriate amount of suspension buffer (0.025M Bis-Tris, 20% glycerol, pH = 7.0), the sample was stored at -80°C for later use, pay attention to extraction The process should always be kept at low temperature to prevent protein degradation. Samples were subjected to blue-green mild gel electrophoresis. The BN-PAGE gel uses a concentration gradient of 3.5% to 12%, and the 3.5% and 12% gels are pre-prepared and placed on ice to prevent gelation. ...

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Abstract

The invention provides a construction method of a soybean seed protein expression profile and an application of the soybean seed protein expression profile to analysis of biological metabolism ways, and belongs to the technical field of protein expression profile construction technologies. According to the construction method, soybean protein electrophoresis films are obtained by adoption of an SDS-PAGE technology; the soybean protein electrophoresis films are graded; and trypsin enzymolysis and liquid chromatography and mass spectrum combination (LC-MS) are respectively carried out on gradedproteins to authenticate the varieties and contents of the soybean proteins. The soybean proteins are graded and authenticated according to electrophoresis, so that benefit is brought to detect low-content proteins and low-richness proteins are prevented from being covered by high-richness proteins, thereby obtaining more proteins. Through grading and authenticating the soybean proteins, functional features of the soybean proteins are found from the angle of bioinformatics, and protein classification and database construction are carried out to provide technical and data support for soybean protein-related researches.

Description

technical field [0001] The invention belongs to the technical field of protein expression profile construction, and in particular relates to a method for constructing soybean seed protein expression profile and the application of soybean protein expression profile in analyzing biological metabolic pathways. Background technique [0002] Seeds are the reproductive system of seed plants, play an important role in the continuation of species, and occupy a central position in the life cycle of plants. Soybean dry seeds are the main edible part of human beings and are also important industrial raw materials. The protein content of soybean seeds accounts for about 40% of the dry weight of the seeds, and is rich in eight essential amino acids needed by the human body. Soybean seed protein is mainly stored in the cotyledons, which has an extremely important impact on seed germination, embryonic development, resistance and antinutrition. [0003] With the development of protein-bas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 邱丽娟张明俊赵宇杨
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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