Method for screening antifungal substance acting on glucosylceramide as target

A technology of ceramide glycolipid and ceramide, applied in the field of antifungal substances, can solve the problems of unclear target and toxicity of antifungal substances, large workload, etc., and achieve the effects of accurate and reliable results, simple screening process and clear targets

Inactive Publication Date: 2014-01-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the conventional plate method is an in vivo screening method, it has a large workload, and the targets and toxicity of the screened antifungal substances are not clear, which requires further clarification through a large number of experiments

Method used

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  • Method for screening antifungal substance acting on glucosylceramide as target
  • Method for screening antifungal substance acting on glucosylceramide as target
  • Method for screening antifungal substance acting on glucosylceramide as target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the construction of two mutant bacterial strains

[0036] 1. Ultraviolet mutagenesis of Aspergillus nidulans wild strain A28

[0037] The spores of Aspergillus nidulans wild bacterial strain A28 were spread onto the MAG solid plate containing 50 μg / ml HSAF, under the ultraviolet lamp (100J / m 2 , the lethal rate was 90%) for mutagenesis treatment.

[0038] 2. Screening and identification of barA gene mutants and gcs1 gene mutants of Aspergillus nidulans

[0039] 1. Place the mutagenized plate at 28°C for 3 days, then pick large colonies and place them on a MAG solid plate containing 50 μg / ml HSAF for further confirmation.

[0040] 2. Using the mutant genomic DNA as a template, PCR amplifies the barA gene (ceramide synthetase gene) and performs sequencing analysis. Using the mutant genomic DNA as a template, the gcs1 gene (glycosylceramide transferase gene) was amplified by PCR and sequenced. If there is a mutation in the barA gene, gcs1 gene, or both th...

Embodiment 2

[0053] Embodiment 2, the preparation of antifungal factor (HSAF)

[0054] Antifungal factor (HSAF): an antifungal substance from Lysobacter enzymogenes C3 that targets ceramide glycolipids (Rittenour, W.R., Chen, M., Cahoon, E.B., and Harris, S.D. (2011 ). Control of glucosylceramide production and morphogenesis by the Bar1 ceramide synthase in Fusarium graminearum. PLoS One 6, e19385. doi: 10.1371 / journal.pone.0019385).

[0055] HSAF was prepared according to the method in the literature (Yu F, Zaleta-Rivera K, Zhu X, Huffman J, Millet JC, et al. (2007) Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action. Antimicr Agents Chemoth 51:64-72.).

Embodiment 3

[0056] Embodiment 3, the inhibitory effect of HSAF on wild strain and mutant strain

[0057] 1. HSAF treatment and morphology observation

[0058] Aspergillus nidulans barA gene mutants were respectively placed in the middle of two solid plates (the first solid plate was a MAG solid plate, and the second solid plate was a MAG solid plate containing 50 μg / ml HSAF), and cultured at 28° C. for 3 days.

[0059] Aspergillus nidulans gcs1 gene mutants were respectively placed between two solid plates (the first solid plate was a MAG solid plate, and the second solid plate was a MAG solid plate containing 50 μg / ml HSAF), and cultured at 28° C. for 3 days.

[0060] Aspergillus nidulans wild strain A28 was placed in the middle of two solid plates (the first solid plate was a MAG solid plate, and the second solid plate was a MAG solid plate containing 50 μg / ml HSAF), and cultured at 28°C for 3 days.

[0061] see results figure 1 with figure 2 . On the first solid plate, there was n...

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Abstract

The invention discloses a method for screening an antifungal substance acting on glucosylceramide as a target. The method provided by the invention includes the following steps: a wild fungus strain and a mutant strain are respectively cultured on solid plates containing a substance to be assayed, and if the growth of the mutant strain on the plate is better than the growth of the wild fungus strain on the plate, the substance to be assayed is a candidate antifungal substance or a candidate substance containing the antifungal substance; and the target of the antifungal substance is glucosylceramide. The method established by the invention has the advantages that: the target is specific, moreover, the method integrates in vivo screening and activity assay, whether the substance is the antifungal substance targeting glucosylceramide can be judged only according to the difference between the sensitivities of the mutant strain and the wide strain on the antifungal substance, the screening process is simpler, and can save time and labor, and the result is more accurate and reliable. The method has a great application prospect in the screening and development of green control agents for fungal diseases.

Description

technical field [0001] The invention relates to a method for screening antifungal substances with ceramide glycolipid as the action target. Background technique [0002] Plant defensins are broad-spectrum antifungal short peptides ubiquitous in a class of plants, which play a key role in plant defense against pathogenic fungal invasion. Among them, the mechanism of the plant defensin RsAFP2 from carrot is relatively clear at present, and the antibacterial effect of RsAFP2 is realized by combining with ceramide glycolipid (glucosylceramide) in the fungal cell membrane. RsAFP2 is not toxic to humans and plants because RsAFP2 cannot bind to ceramide glycolipids in humans and plants. Therefore, the development of pesticides whose mechanism of action is similar to plant defensin RsAFP2 is an ideal way to achieve green control of plant fungal diseases. [0003] The large-scale expression of plant defensins by using microorganisms is an ideal model to realize their large-scale ap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/18C12N1/14C12R1/66
Inventor 李少杰孙宪昀张振颖张晗星
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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