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Method for constructing target protein interaction network based on high-throughput sequencing

A target protein, high-throughput technology, applied in the field of target protein interaction network construction based on high-throughput sequencing, can solve the problems of limited, labor-intensive, time-consuming and labor-intensive detection capabilities.

Active Publication Date: 2021-02-02
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional yeast two-hybrid screening library system still has deficiencies, such as the limitation of detection ability, labor-intensive and time-consuming operation screening process, unstable quality of results obtained in a single operation, and limited number of target proteins. protein, etc.
[0004] In summary, yeast two-hybrid technology is very important for the study of plant protein functions without a reference genome, but the existing library screening method is time-consuming and labor-intensive and has limited detection capabilities. The number of clones is limited, and a new method for yeast two-hybrid screening library to find all interacting proteins of the target protein in a single library as much as possible

Method used

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  • Method for constructing target protein interaction network based on high-throughput sequencing
  • Method for constructing target protein interaction network based on high-throughput sequencing
  • Method for constructing target protein interaction network based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1. Screen library conversion process

[0044]In this example, the species of the target protein is chrysanthemum. Chrysanthemum CmMPK3 kinase as the target protein, CmMPK3 kinase gene (genbank: MG334203). The known target protein was constructed on the pGBKT7 vector by conventional methods to obtain the BD-bait pGBKT7-CmMPK3, and the yeast AD library was used as the prey library; pGBKT7-CmMPK3 (5 μg) was co-transformed with the yeast AD library (10 μg) using Matchmaker TM These plasmids were co-transformed into the Y2H yeast strain using the Gold Yeast Two-Hybrid System (Clontech).

Embodiment 2 4

[0045] Example 2. Screening of interacting proteins in four-deficiency medium

[0046] Transfer the transformed yeast liquid to 3mL YPDplus (Huayueyang PPT12) medium, culture at 30°C for 4h, centrifuge at 2000rpm for 5min, discard the supernatant, add 1mL sterile water to suspend, and obtain the bacterial liquid; take the obtained 100 μL of bacterial solution was added to 10 mL of four-deficient medium SD / -Leu / -Trp / -His / -Ade liquid medium (Huayueyang *TP0239) at 30°C and 200 rpm for 24h, 72h, and 80h, respectively, and samples were taken.

Embodiment 3

[0047] Embodiment 3.PCR amplification

[0048] The bacterial liquid sampled at 24h, 72h, and 80h was used as a template, and AD-F (SEQ ID NO.1) and AD-R (SEQ ID NO.2) were used as primers to carry out MightyAmp (TaKaRa) PCR amplification according to the procedure shown below. Each sample had 5 repetitions, and the PCR products of the bacterial liquid samples in 24h, 72h, and 80h were obtained respectively.

[0049] PCR program:

[0050]

[0051] The PCR system is:

[0052]

[0053] Agarose gel electrophoresis was used to detect the enrichment of the target protein in the PCR products of the bacterial liquid samples in the three time periods ( figure 1 ), figure 1 It shows that, taking the 5000bp marker as a reference, it is found that the bands are scattered in the range of 500bp-5000bp at 24h and there is no obvious single band, and the length of YPD plus culture has no obvious impact on the results; while at 72h, the PCR amplification product narrows the range to ...

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Abstract

The invention discloses a method for constructing a target protein interaction network based on high-throughput sequencing. According to the method, a BD-bait and a prey library are co-transformed into a Y2H yeast strain, then culturing in a YPDplus culture medium at 30 DEG C for 4 hours, discarding the supernatant, adding 1mL of sterile water medium for suspension, taking 100muL of suspension bacterial liquid and adding into 10ml of SD / -Leu / -Trp / -His / -Ade liquid culture medium for shaking culture, sampling respectively after shaking culture for 24 hours, 72 hours and 80 hours, the sampled bacterial liquids are used as templates for PCR (Polymerase Chain Reaction) amplification, carrying out agarose gel electrophoresis to test the screening degree of the bacterial liquids, recycling products, carrying out high-throughput sequencing, carrying out gene abundance and difference analysis after de novo assembly, and taking a difference gene as a protein coding gene interacting with a targetgene in the library, so as to preliminarily construct an interaction network of the target protein. The method is also suitable for constructing a target protein interaction network of species without reference genes. The method simplifies the steps of a yeast two-hybrid screening library, and the method is not limited by the number of clones screened once, and can find all interaction proteins of a target protein in a single library.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing a target protein interaction network based on high-throughput sequencing. [0002] technical background [0003] Yeast two-hybrid is a system consisting of two structural domains that are separated from each other but necessary for function. It was proposed by Fields and Song in 1989 and is a classic experiment for screening interacting proteins. Yeast two-hybrid technology has many advantages in research. For example, it uses eukaryotic cells as hosts, which can restore the interaction between proteins under physiological conditions to the greatest extent. Since the reaction is carried out through the physiological metabolic process, many weak interactions that are difficult to detect can be revealed by biological amplification, and the sensitivity of this method is high. However, the traditional yeast two-hybrid screening library system still has def...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12N15/10G16B5/00G16B25/20
CPCC12Q1/6869C12N15/1055G16B5/00G16B25/20C12Q2531/113C12Q2535/122Y02A50/30
Inventor 宋爱萍余琪胡月姮张璐瑶陈素梅管志勇房伟民陈发棣
Owner NANJING AGRICULTURAL UNIVERSITY
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