Construction method for single knock-out mutant bacterial strain producing deoxynivalenol in high-yield mode

A technology for deoxynivalenol and construction methods, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, enzymes, etc., can solve the problem of difficult screening of high-yielding strains, Fusarium graminearum There is little difference in toxin production ability, etc., to achieve high toxin production and reduce adverse consequences

Inactive Publication Date: 2016-05-11
NORTHWEST A & F UNIV
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  • Claims
  • Application Information

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Problems solved by technology

However, the toxin-producing ability of Fusarium graminearum obtained in nature has li...

Method used

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  • Construction method for single knock-out mutant bacterial strain producing deoxynivalenol in high-yield mode
  • Construction method for single knock-out mutant bacterial strain producing deoxynivalenol in high-yield mode
  • Construction method for single knock-out mutant bacterial strain producing deoxynivalenol in high-yield mode

Examples

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Embodiment 1

[0023] Example 1: Construction of knockout mutant strains with high-yield deoxynivalenol

[0024] (1) Using the DNA of Fusarium graminearum wild-type PH-1 strain as a template, using primers PDE2-1F+PDE2-2R, PDE2-3F+PDE2-4R to amplify the upstream U of the start codon and downstream of the stop codon of the gene Sequence D; using the plasmid PFL2 containing neomycin resistance gene as a template, using primers GEN / F+GE / R, FN / F+GEN / R to amplify neomycin resistance gene sequences G1 and G2; among them, PCR Amplification reaction system: 50 μl PCR reaction solution, containing 50 ng template DNA, 10 μl 5XPfu buffer, 1 μl 10 mmol dNTP, 0.5 μl primer P1 (10 μmol), 0.5 μl Primer P2 (10 micromolar), 0.4 microliter FastPfuDNA polymerase (5 activity units / microliter); PCR reaction conditions: 94℃ for 2 minutes; 94℃ for 20 seconds, 55℃ for 20 seconds, 72℃ for 40 seconds, 32 units Cycle; and final extension at 72°C for 5 minutes; after the reaction is completed, electrophoresis on a 1% aga...

Embodiment 2

[0028] Example 2: Evaluation of toxin synthesis of genetically modified mutants

[0029] 1. Liquid culture toxin production method: Obtain wild-type and mutant spores and add them to liquid toxin production medium (liquid toxin production medium (1 liter): 30 g sucrose, 1 g sodium nitrate, 1 g ammonium dihydrogen phosphate) , 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 10mg ferrous sulfate heptahydrate, 0.03% vegetable gel and 200 microliters of trace element mixture (trace element mixture (100ml): 5g citric acid, 5 G zinc sulfate heptahydrate, 0.25 g copper sulfate pentahydrate, 50 mg manganese sulfate monohydrate, 50 mg boric acid, 50 mg sodium molybdate dihydrate.), the pH value is adjusted to 6.5 with sodium hydroxide.) to a final concentration of 10 4 Spores / ml, stand in the dark and cultivate for 7 days to determine the toxin in the medium. Toxin determination uses the ELISA kit produced by Beacon. The specific operation is: suck the culture medium, centri...

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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 present invention is a method for constructing a knockout mutant strain of high-yield deoxynivalenol, and specifically relates to a method for constructing a knockout mutant strain of high-yielding deoxynivalenol of Fusarium graminearum. Background technique [0002] Fusarium graminearum is a pathogenic fungus that causes wheat head blight (Fusariumheadblightorscab). It not only seriously affects crop yields, but also produces a variety of mycotoxins harmful to humans and animals, deoxynivalenol (DON), which can make food or feed The quality is reduced. Deoxynivalenol (DON) is a sesquiterpene compound that is not easily degraded and has high stability. It exists widely in nature. It is considered to be mainly synthesized by Fusarium in the absence of nutrients. The main natural toxins are found in wheat with head blight disease. DON toxin is one of the fungal toxins with the highest pollution rate. It mainly pollutes cereal crops such as wheat and cor...

Claims

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Application Information

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IPC IPC(8): C12N15/80
CPCC12N9/16C12N15/80C12Y301/04001
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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