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Recombinant vector, host cell and application of ustilaginoidea virens effector protein

A rice smut fungus and effector protein technology, applied in application, bacteria, recombinant DNA technology and other directions, can solve problems such as polluting the environment and endangering human health, and achieve the effects of improving disease resistance, improving plant resistance and broad application prospects.

Inactive Publication Date: 2020-03-17
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Aiming at the disease resistance of plants, at present, chemical methods such as spraying pesticides are generally used to prevent and control the disease. This method will pollute the environment and endanger human health.

Method used

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  • Recombinant vector, host cell and application of ustilaginoidea virens effector protein
  • Recombinant vector, host cell and application of ustilaginoidea virens effector protein
  • Recombinant vector, host cell and application of ustilaginoidea virens effector protein

Examples

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Effect test

Embodiment 1

[0024] This embodiment provides a recombinant vector and a host cell containing the recombinant vector. The recombinant vector and the host cell are used to express the effector protein of Aspergillus oryzae; The coding gene is inserted between the EcoR I and Xho I restriction sites of the pET-32a vector, and the map of the pET-32a vector is attached figure 1 Shown; The rice smut effect protein is called UV_3696, and its amino acid sequence is shown in the sequence listing SEQ ID NO: 1; the nucleotide sequence of the coding gene of the rice false smut effect protein is as the sequence listing SEQ ID NO: 2.

[0025] In addition, the host cells containing the above-mentioned recombinant vectors are obtained through the following steps:

[0026] (1) Take 50 μL of Escherichia coli BL21 competent cell suspension from the -80°C refrigerator, thaw it at room temperature, and put it on ice immediately for later use.

[0027] (2) Add 5 μL of the above-mentioned recombinant vector to ...

Embodiment 2

[0031] This embodiment provides a method for expressing and purifying the effector protein of rice smut, which comprises the following steps:

[0032] (1) Place 1 mL of the host cells containing the recombinant vector provided in Example 1 above in 5 mL of LB liquid medium, and add antibiotics (ampicillin), then place at 37° C. and 200 rpm for overnight shake flask culture, Obtain culture medium.

[0033] (2) Add all the above-mentioned culture solution to 100mL LB liquid medium, add ampicillin, and then place it at 37°C and 200rpm for expanded culture until OD600=0.6, then add 100μL with a concentration of 0.1mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG), and placed under the conditions of 23°C and 140rpm to induce expression for 4h, to obtain the induced bacterial liquid.

[0034] (3) Put all the above-mentioned induced bacteria liquid into a 250mL centrifuge bottle, centrifuge at 10000rpm for 5min, discard the supernatant, resuspend the bacteria with protein dissolutio...

Embodiment 3

[0039] This embodiment provides an application of the rice smut effect protein solution (UV_3696) obtained in the above-mentioned embodiment 2 in increasing the callose content of Arabidopsis thaliana. Specifically, the method includes the following steps: take the 1 μm concentration of the above-mentioned effector protein solution of Aspergillus oryzae which has been diluted to 0.01 μmol / L, and evenly spray it on the leaves of Arabidopsis thaliana, and store it for 24 hours for later use.

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Abstract

The invention discloses a recombinant vector, a host cell and an application of an ustilaginoidea virens effector protein, and belongs to the technical field of biology. The recombinant vector is usedfor expressing an ustilaginoidea virens effect protein, and is obtained by inserting a coding gene of the ustilaginoidea virens effect protein to a position between EcoR I and Xho I restriction enzyme cutting sites of a pET-32a vector. The amino acid sequence of the ustilaginoidea virens effect protein is shown in the formula of SEQ ID NO: 1 in a sequence table and the nucleotide sequence of thecoding gene of the ustilaginoidea virens effect protein is shown in the formula of SEQ ID NO: 2 in the sequence table. According to the invention, the ustilaginoidea virens effect protein obtained byin vitro expression is sprayed on arabidopsis thaliana leaves so that the contents of active oxygen and callose in the arabidopsis thaliana can be obviously improved and the disease resistance of thearabidopsis thaliana to pathogenic bacteria Pseudomonas syringae pv.tomato (Pst) DC3000 can be improved. A novel way is provided for improving the plant resistance and inducing the plant defense reaction, and the recombinant vector has a wide application prospect in agricultural production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a recombinant vector, a host cell and an effector protein of rice smut bacteria. Background technique [0002] For the disease resistance of plants, chemical methods such as spraying pesticides are generally used to prevent and control the disease. This method will pollute the environment and endanger human health. [0003] Therefore, there is an urgent need for a natural material that is environmentally friendly and can improve plant disease resistance to solve the above problems. Contents of the invention [0004] The purpose of the present invention is to provide a recombinant vector to solve the problems raised in the background art above. [0005] In order to achieve the above purpose, embodiments of the present invention provide the following technical solutions: [0006] A kind of recombination vector, described recombination vector is used for expressin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/31C12N1/21C12N15/82C07K14/37A01H5/00A01H6/20C12R1/19
CPCC07K14/37C12N15/70C12N15/8242C12N15/8281
Inventor 李帅魏松红向世博周建铭邢帆王应玲海樱凡
Owner SHENYANG AGRI UNIV
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