A method for constructing a high-yield deoxynivalenol knockout mutant strain

A technology of deoxynivalenol and a construction method, which is applied in the direction of using a vector to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problem that the toxin production capacity of Fusarium graminearum is not very different, and it is difficult to screen out high toxin production. strains, etc., to achieve the effect of high toxin production and reduction of adverse consequences

Inactive Publication Date: 2019-10-22
NORTHWEST A & F UNIV
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Problems solved by technology

However, the toxin-producing ability of Fusarium graminearum obtained in nature has little difference, and it is difficult to screen high-yield strains for large-scale production

Method used

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  • A method for constructing a high-yield deoxynivalenol knockout mutant strain
  • A method for constructing a high-yield deoxynivalenol knockout mutant strain
  • A method for constructing a high-yield deoxynivalenol knockout mutant strain

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Embodiment 1

[0023] Example 1: Construction of a knockout mutant strain with high yield of deoxynivalenol

[0024] (1) Using the DNA of Fusarium graminearum wild-type PH-1 strain as a template, use primers PDE2-1F+PDE2-2R, PDE2-3F+PDE2-4R to amplify the upstream U of the gene start codon and the downstream of the stop codon Sequence D; using the plasmid PFL2 containing the neomycin resistance gene as a template, using primers GEN / F+GE / R, FN / F+GEN / R to amplify the neomycin resistance gene sequences G1 and G2; wherein, PCR Amplification reaction system: In 50 microliters of PCR reaction solution, containing 50 nanograms of template DNA, 10 microliters of 5X Pfu buffer, 1 microliter of 10 millimolar dNTPs, 0.5 microliters of primer P1 (10 micromolar), 0.5 microliters liter of primer P2 (10 micromole), 0.4 microliter of FastPfu DNA polymerase (5 activity units / microliter); PCR reaction conditions: 94°C for 2 minutes; 94°C for 20 seconds, 55°C for 20 seconds, 72°C for 40 seconds, 32 cycles; an...

Embodiment 2

[0028]Example 2: Evaluation of Toxin Synthesis Amount of Genetically Modified Mutants

[0029] 1. Liquid culture toxin production method: obtain wild-type and mutant spores, add to liquid toxin production medium (liquid toxin production medium (1 liter): 30 grams of sucrose, 1 gram of sodium nitrate, 1 gram of ammonium dihydrogen phosphate , 0.5 gram of magnesium sulfate heptahydrate, 0.5 gram of potassium chloride, 10 milligrams of ferrous sulfate heptahydrate, 0.03% vegetable gel and 200 microliters of trace element mixed solution (trace element mixed solution (100 milliliters): 5g citric acid, 5 gram of zinc sulfate heptahydrate, 0.25 gram of copper sulfate pentahydrate, 50 milligrams of manganese sulfate monohydrate, 50 milligrams of boric acid, 50 milligrams of sodium molybdate dihydrate.), the pH value is adjusted to 6.5 with sodium hydroxide.) to a final concentration of 10 4 spores / ml, cultured in the dark for 7 days, and the toxin in the medium was determined. Toxin ...

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Abstract

A construction method for a single knock-out mutant bacterial strain producing deoxynivalenol in a high-yield mode includes the following steps of firstly, knocking a negative regulatory factor high-affinity phosphodiesterase gene FgPDE2 generated in the fusarium graminearumout deoxynivalenol synthesis process out of a genome of fusarium graminearum PH-1 through a split-PCR method, and obtaining the single knock-out mutant, wherein the gene FgPDE2 and upstream and downstream sequences are shown in SEQ ID NO:1; secondly, PEG-mediated protoplast is converted into fusarium graminearum, knocking out converters through resistance screening and marker screening, and verifying that the target gene knocking out the converters is replaced with a neomycin resistance gene through primers 5F and 6R with the genome of fusarium graminearum serving as the reference. The toxicity production amount of the single knock-out mutant bacterial strain is greatly increased; the toxicity production amount of a double knock-out mutant bacterial strain is higher; the gene modification mutant loses the capacity for infecting wheatears, and negative effects caused by possible diffusion of the double knock-out mutant bacterial strain are greatly reduced.

Description

technical field [0001] The invention discloses a method for constructing a high-yield deoxynivalenol knockout mutant strain, in particular to a construction method for a high-yield Fusarium graminearum deoxynivalenol knockout mutant strain. Background technique [0002] Fusarium graminearum is the pathogenic fungus that causes Fusarium head blight or scab in wheat. It not only seriously affects crop yield, but also can produce a variety of mycotoxins deoxynivalenol (DON) that are harmful to humans and animals. Decreased quality of food or feed. Deoxynivalenol (deoxynivalenol, DON) is a sesquiterpene compound, which is not easy to degrade and has high stability. The main natural toxin is present in head blight-affected wheat. DON toxin is one of the mycotoxins with the highest pollution rate. It mainly contaminates cereal crops such as wheat and corn, and also contaminates food products, such as bread, biscuits, wheat-based snacks, etc. In addition, DON residues are also re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80
Inventor 江聪刘慧泉许金荣王晨芳王建华张世杰陈代朋吴春兰
Owner NORTHWEST A & F UNIV
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