Method for extracting polyribosomes from rice based on immunoprecipitation

A polyribosome and immunoprecipitation technology, applied in the field of molecular biology, can solve the problems of complex operation process, high requirements on experimental equipment and low product yield.

Inactive Publication Date: 2016-08-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention aims at the above-mentioned deficiency that prior art exists, proposes a kind of method based on immunoprecipitation method to extract polyribosome from rice, to solve the high requirement of experimental equipment when extracting polyribosome in the past, complicated operation process, And the problem of low product yield

Method used

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  • Method for extracting polyribosomes from rice based on immunoprecipitation
  • Method for extracting polyribosomes from rice based on immunoprecipitation
  • Method for extracting polyribosomes from rice based on immunoprecipitation

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Experimental program
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Effect test

Embodiment 1

[0037] Construction of OsRPL18B1 (Os03g0341100) and OsRPL18B2 (Os05g0155100) plant expression vectors fused with Flag tags. There are 6 genes encoding RPL18 protein in rice, of which 3 belong to RPL18A and the other 3 belong to RPL18B. The present invention selects OsRPL18B1 (Os03g0341100) , OsRPL18B2 (Os05g0155100) two genes added tags, further experiments ( figure 1 ).

[0038] We constructed plant expression vectors of OsRPL18B1 (Os03g0341100) and OsRPL18B2 (Os05g0155100) fused with Flag tags, and transformed them into rice Nipponbare respectively, so that the fusion proteins were overexpressed under the promoter of 35S promoter and Ω-HF enhancer ( figure 2 ).

Embodiment 2

[0039]Molecular identification of embodiment 2 transgenic strains

[0040] Since we need to obtain as many tagged ribosome-mRNA complexes as possible in the transgenic lines, GUS staining and western blot identification were performed on the obtained transgenic lines, and the lines with higher expression of fusion protein were selected for further analysis. further research.

[0041] Stained by GUS ( image 3 ) and western blot ( Figure 4 ) identification, we selected four strains B1-13, B1-29, B2-26, B2-38 for the next experiment.

Embodiment 3

[0042] The extraction of embodiment 3 polyribosomes

[0043] Ribosomes can remain bound to mRNA in the presence of high magnesium ions, so EGTA is used to chelate other metal ions in tissue homogenate; adding chloramphenicol and cycloheximide can also inhibit the dissociation of ribosomes and mRNA .

[0044] All experimental equipment must be free from RNase contamination. According to different materials, RNase can be removed by drying at 160°C for 4 hours, soaking in 0.1% DEPC or 10% hydrogen peroxide and autoclaving. Non-alcoholic solutions need to be prepared with RNase free milliQ water. In the experiment, try to keep the operation on ice or at 4°C.

[0045] 1) Fragmentation of plant tissue

[0046] In the following experiments, 1% volume of 0.1M PMSF was added to the current system every half hour;

[0047] Add twice the volume of PEB to the liquid nitrogen ground material powder and co-grind with liquid nitrogen (at least 2ml sample). The glass homogenizer in the o...

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Abstract

The invention belongs to the technical field of molecular biology, and in particular relates to a method for extracting polysome from rice on the basis of immune-precipitation. The method comprises the steps: by mainly adopting an immunization method, enriching a Flag tag-labeled ribosome-mRAN (messenger ribonucleic acid) compound by using affinity chromatography, chelating other metal ions in tissue homogenate by using EGTA (ethylene glycol tetraacetic acid), and inhibiting the dissociation of ribosome and mRNA by adding chloramphenicol and cycloheximide, wherein the formulas of an extraction solution and an elution solution as well as the material homogenate and concentration modes are improved; the ribosome-mRAN compound is eluted by using EDTA with the final concentration of 0.1M. The method disclosed by the invention is expected to separate 2-4 important new functional genes involving in low-temperature stress, ensures the normal growth and proliferation of plants under the low-temperature stress, and finally plays an important role in yield development.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for extracting polyribosomes from rice. Background technique [0002] Polyribosome refers to a bead-like structure formed by multiple or even dozens of ribosomes attached to an mRNA molecule in series when protein is synthesized. When synthesizing multiple proteins, ribosomes do not work alone, and often exist in the form of polyribosomes. At the start codon of the mRNA, the ribosomal subunits assemble into a complete initiation complex, which then moves toward the 3' end of the mRNA until it reaches the stop codon. When the first ribosome leaves the start codon, the position of the vacated start codon is sufficient for another ribosome to bind, and the small subunit of the second ribosome joins and assembles into a complete Initiation complex, which begins protein synthesis. Similarly, the third ribosome and the fourth ribosome, in turn, bind to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 明凤王尧峰奚丹丹罗莉琼沈佳斌
Owner FUDAN UNIV
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