Method for acquiring two-dimensional strawberry electrophoresis differential protein map
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A protein map and two-dimensional electrophoresis technology, which is applied in the field of obtaining the differential protein map of strawberry two-dimensional electrophoresis, can solve the problems of strawberry research blank, unsuitable strawberry, etc.
Inactive Publication Date: 2013-08-28
HANGZHOU ACAD OF AGRI SCI
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[0004] At present, the method of using two-dimensional electrophoresis to obtain protein maps has a certain degree of application in the field of biomedicine and some plant research (such as apple leaves), but it is almost blank in strawberry research.
Zhang Caixia et al obtained a high-quality protein spot map in apple leaves by means of phenol extraction, but this method is not suitable for strawberry
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Embodiment 1
[0058] Embodiment 1, a method for obtaining the differential protein map of strawberry two-dimensional electrophoresis, the strawberry variety used is "Red Cheek", and its leaves are taken; the following steps are carried out in sequence:
[0059] 1), protein extraction:
[0060] Include the following steps:
[0061] ①. Take 1 g of strawberry leaves and add 4 ml of liquid nitrogen, after grinding, suspend it in 15 ml of 10% (v / v) trichloroacetic acid (prepared in acetone) solution, and precipitate at -20°C for 12 hours;
[0062] ②. Centrifuge the product obtained in step ① (4°C, 15000r / min, 30min), and discard the supernatant;
[0063] ③. Suspend the precipitate obtained in step ② in 15ml of B-mercaptoethanol cold acetone solution; centrifuge (4°C, 15000r / min, 30min), repeat the suspension and centrifugation of the obtained precipitate 5 times;
[0064] The preparation method of the B-mercaptoethanol cold acetone solution is: mixing cold acetone and B-mercaptoethanol accordi...
Embodiment 2
[0090] Embodiment 2, with the blade of strawberry variety " Sweet Charlie ", replace the blade of " red cheek " strawberry in embodiment 1, all the other are the same as embodiment 1.
[0091] The final spectrum obtained is as figure 2 shown. according to figure 2 , we can draw the following conclusions: for the leaves of different strawberry varieties, the method of the present invention can also stably obtain high-quality protein expression maps. The protein map is clear, the number of protein spots is large, and the background is very clear.
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Abstract
The invention discloses a method for acquiring a two-dimensional strawberry electrophoresis differential protein map. The method comprises the following steps: (1) extracting proteins; (2) performing first-dimensional IPG isoelectric focusing electrophoresis; (3) balancing an adhesive tape; (4) transferring the adhesive tape and performing SDS-polyacrylamide gel electrophoresis; and (5) performing gel scanning and image analysis. An inventor considers the characteristic that strawberry leaves are rich in polysaccharide polyphenol substances in the invention process, the strawberry leaf protein is subjected to repeated precipitation extraction by virtue of acetone, the lysate formula is improved, the centrifugal rate, the cleaning frequency of crude protein precipitations and concentration of urea in lysate are improved, and a set of differential two-dimensional electrophoresis proteomics method suitable for strawberry leaves is constructed, so that the high-quality strawberry protein differential map is obtained. The method can be used for obtaining the two-dimensional strawberry leaf protein electrophoresis gel map with clear protein points, a large number of proteins, uniform protein distribution and a clear background.
Description
technical field [0001] The invention relates to a method for obtaining a differential protein map of strawberry two-dimensional electrophoresis. Background technique [0002] Strawberry is an important economic crop. Using two-dimensional electrophoresis-based proteomics technology, we can quickly mine the differential gene expression products produced in strawberry in response to pathogen infection, so as to elucidate the disease resistance mechanism and provide guidance for future strawberry disease resistance breeding. The work provides important technical support. [0003] Existing methods for studying the mechanism of strawberry disease resistance often rely on genomics technology, which can excavate the important genes of plants responding to stress to a certain extent. But because protein is the final product of gene expression, finding out the direct differential protein related to disease resistance will be more helpful to the research of disease resistance mechani...
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