CN102586394B - Method for screening antifungal substance acting on glucosylceramide as target - Google Patents
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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
The present invention relates to a kind of screening and take the method for the antifungal substance that the ceramide glycolipid is action target.
Background technology
Plant alexin (plant defensin) is ubiquitous broad-spectrum antifungal small peptide in a class plant, plant is resisted to the pathogenic fungi invasion and play keying action.Wherein the plant alexin RsAFP2 mechanism from Radix Dauci Sativae is relatively clear at present, the RsAFP2 bacteriostatic action be by with fungal cell membrane in ceramide glycolipid (glucosylceramide) in conjunction with realizing.RsAFP2 does not have toxicity to people and plant, and this is because RsAFP2 can not be combined with the ceramide glycolipid of people and plant.Therefore, the agricultural chemicals that the research and development mechanism of action is similar to plant alexin RsAFP2 is the desirable approach that realizes the fungal diseases of plants green prevention and control.
Utilizing microorganism great expression plant alexin is the idealized model that realizes its mass-producing application, yet, because the synthesis mode of plant alexin is special, great expression is difficult to realize, has seriously limited its application on agricultural.Therefore, the antifungal substance that the target in other sources such as screening microorganism is the ceramide glycolipid will be more effective and feasible approach, and there is no at present the report of specificity screening method.
The screening method of antifungal substance is mainly flat band method at present.Although conventional flat band method is a kind of Vivo Studies on Screening method, target and the toxicity of the antifungal substance that its workload is large, screen are all unclear, and needs further be illustrated by great many of experiments.
Summary of the invention
The purpose of this invention is to provide a kind of screening and take the method for the antifungal substance that the ceramide glycolipid is action target
The method of the antifungal substance that Screening target provided by the invention is the ceramide glycolipid, comprise the steps A: wild fungus bacterial strain and mutant strain are cultivated containing on the solid plate of test substance respectively, if the growing state of mutant strain on flat board is better than the growing state of wild fungus bacterial strain on flat board, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance; The target of described antifungal substance is the ceramide glycolipid; Described mutant strain is for to carry out by the ceramide glycolipid metabolism approach genes involved of described wild fungus bacterial strain the bacterial strain that afunction obtains.
The method of the antifungal substance that described Screening target is the ceramide glycolipid also can comprise the steps B and/or step C:
Step B: the spore of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if in the mycelia branch containing in the liquid nutrient medium of described test substance more than not containing the mycelia branch in the liquid nutrient medium of described test substance, the antifungal substance that described test substance be the candidate or candidate's the material that contains antifungal substance;
Step C: the mycelia of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if in the mycelia branch containing in the liquid nutrient medium of described test substance more than not containing the mycelia branch in the liquid nutrient medium of described test substance, the antifungal substance that described test substance be the candidate or candidate's the material that contains antifungal substance.
In described steps A, the ceramide glucoside transferase gene shown in the sequence 2 of the ceramide synthase gene shown in the sequence 1 that described ceramide glycolipid metabolism approach genes involved is sequence table and/or sequence table.In described steps A, described wild fungus bacterial strain is Aspergillus nidulans (Aspergillus nidulans) wild strain A28.In described steps A, described mutant strain is Aspergillus nidulans (Aspergillus nidulans) UV-1 or Aspergillus nidulans (Aspergillus nidulans) UV-13.
Aspergillus nidulans (Aspergillus nidulans) UV-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 27th, 2012 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5806.Aspergillus nidulans (Aspergillus nidulans) UV-1 claim again Aspergillus nidulans barA gene mutation body.
Aspergillus nidulans (Aspergillus nidulans) UV-13 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 27th, 2012 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5807.Aspergillus nidulans (Aspergillus nidulans) UV-13 claim again Aspergillus nidulans gcs1 gene mutation body.
In described steps A, described culture condition can be 28 ℃ and cultivates 3d.In above steps A, described solid plate specifically can be the MAG solid plate.In described step B, described culture condition can be 28 ℃ and cultivates 8-12h.In described step C, described culture condition can be 28 ℃ and cultivates 3-6h.In described step B and/or described step C, described liquid nutrient medium specifically can be the MAG liquid nutrient medium.Described test substance can be microorganism extracts, plant milk extract or Chinese medical extract etc., also can be compound.
The present invention also provides a kind of and has identified that whether test substance is the method for the target antifungal substance that is the ceramide glycolipid, comprise the steps D: wild fungus bacterial strain and mutant strain are cultivated containing on the solid plate of test substance respectively, if the growing state of mutant strain on flat board is better than the growing state of wild fungus bacterial strain on flat board, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance, if growing state and the wild fungus bacterial strain growing state on flat board of mutant strain on flat board do not have significant difference, the non-antifungal substance that described test substance is the candidate or candidate not containing the material of antifungal substance, the target of described antifungal substance is the ceramide glycolipid, described mutant strain is for to carry out by the ceramide glycolipid metabolism approach genes involved of described wild fungus bacterial strain the bacterial strain that afunction obtains.
Describedly identify that whether test substance is that the method for the target antifungal substance that is the ceramide glycolipid also can comprise the steps E and/or step F:
Step e: the spore of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if the mycelia branch in containing the liquid nutrient medium of described test substance is more than the mycelia branch in not containing the liquid nutrient medium of described test substance, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance, if there is no significant difference in the mycelia branch containing in the liquid nutrient medium of described test substance and the mycelia branch in not containing the liquid nutrient medium of described test substance, the non-antifungal substance that described test substance is the candidate or candidate not containing the material of antifungal substance,
Step F: the mycelia of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if the mycelia branch in containing the liquid nutrient medium of described test substance is more than the mycelia branch in not containing the liquid nutrient medium of described test substance, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance, if there is no significant difference in the mycelia branch containing in the liquid nutrient medium of described test substance and the mycelia branch in not containing the liquid nutrient medium of described test substance, the non-antifungal substance that described test substance is the candidate or candidate not containing the material of antifungal substance.
In described step D, the ceramide glucoside transferase gene shown in the sequence 2 of the ceramide synthase gene shown in the sequence 1 that described ceramide glycolipid metabolism approach genes involved is sequence table and/or sequence table.In described step D, described wild fungus bacterial strain is Aspergillus nidulans (Aspergillus nidulans) wild strain A28.In described step D, described mutant strain is Aspergillus nidulans (Aspergillus nidulans) UV-1 or Aspergillus nidulans (Aspergillus nidulans) UV-13.
In described step D, described culture condition can be 28 ℃ and cultivates 3d.In described step D, described solid plate specifically can be the MAG solid plate.In described step e, described culture condition can be 28 ℃ and cultivates 8-12h.In described step F, described culture condition can be 28 ℃ and cultivates 3-6h.In described step e and/or described step F, described liquid nutrient medium specifically can be the described test substance of MAG liquid nutrient medium and can be microorganism extracts, plant milk extract or Chinese medical extract etc., also can be compound.
The present invention also protects Aspergillus nidulans (Aspergillus nidulans) UV-1, and its deposit number is CGMCC No.5806.
The present invention also protects Aspergillus nidulans (Aspergillus nidulans) UV-13, and its deposit number is CGMCC No.5807.
The present invention also protects Aspergillus nidulans UV-1 and/or the application of Aspergillus nidulans UV-13 in the screening antifungal substance; The target of described antifungal substance is the ceramide glycolipid.
The present invention also protects ceramide synthetase-coding gene and/or the application of ceramide glucoside transferase gene in the screening antifungal substance of fungi; The target of described antifungal substance is the ceramide glycolipid.Described ceramide glycolipid metabolism approach genes involved specifically can be the ceramide glucoside transferase gene shown in the sequence 2 of the ceramide synthase gene shown in the sequence 1 of sequence table and/or sequence table.Described fungi specifically can be Aspergillus nidulans wild strain A28.
The principle of institute of the present invention foundation is as follows: the mechanism of action of the antifungal substance that the targets such as plant alexin RsAFP2 are the ceramide glycolipid be by with fungal cell membrane in ceramide glycolipid (glucosylceramide) in conjunction with realizing.Fungi exists two kinds to participate in the synthetic enzyme of ceramide glycolipid: ceramide synthetic enzyme and ceramide glucoside transferring enzyme.Therefore, arbitrary or disappearance repertoire of the above-mentioned two kinds of enzymes in fungi all can cause ceramide glycolipid content in the mutant cells film reduce or lose, thereby to Reduced susceptibility or the forfeiture of this class antifungal substance.In addition, the antifungal substance that target is the ceramide glycolipid also has the characteristics that cause the excessive bifurcated of hypha,hyphae, can be used as the method for further checking.
Not only target is clear and definite for the method that the present invention sets up, and Vivo Studies on Screening and active the detection are integrated in one, only according to mutant strain and wild strain, to the sensitivity differences of antifungal substance, just can judge whether it is the antifungal substance that target is the ceramide glycolipid, screening process is simpler, time saving and energy saving, and result more accurately and reliably.The present invention has huge application prospect for the screening of the green prevention and control medicine of fungal disease with research and development.
The accompanying drawing explanation
Fig. 1 is the restraining effect of HSAF to Aspergillus nidulans wild strain A28 and Aspergillus nidulans barA gene mutation body.
Fig. 2 is the restraining effect of HSAF to Aspergillus nidulans wild strain A28 and Aspergillus nidulans gcs1 gene mutation body.
The morphological specificity of spore microscopy after HSAF coerces that Fig. 3 is Aspergillus nidulans wild strain A28.
Fig. 4 is active substance HB-2 and the HB-3 growth effect to Aspergillus nidulans wild type strain A28 and Aspergillus nidulans barA gene mutation body.
Fig. 5 is active substance HB-2 and the HB-3 growth effect to Aspergillus nidulans wild type strain A28 and Aspergillus nidulans gcs1 gene mutation body
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.
Aspergillus nidulans (Aspergillus nidulans) wild strain A28, purchased from U.S. genetic of fungi learn the preservation center (Fungal Genetics Stock Center, be called for short FGSC, network address:
www.fgsc.net), preserving number is FGSC #A28 (Genotype:pabaA6, biA1).
MAG liquid nutrient medium: get 20g Fructus Hordei Germinatus extract, 20g glucose, 2g peptone, 1ml trace element mixed solution and 2ml vitamin mixture, with distilled water, dissolve and be settled to 1L.The preparation method of trace element mixed solution: successively by 2.2g ZnSO
47H
2o, 1.1g H
3bO
3, 0.5g MnCl
24H
2o, 0.5g FeSO
47H
2o, 0.17g CoCl
26H
2o, 0.16g CuSO
45H
2o, 0.15g Na
2moO
42H
2o and 05g Na
4-EDTA dissolves with distilled water and is settled to 100ml (with KOH, adjusting pH to 6.5).The preparation method of vitamin mixture: get 100mg vitamin H (vitamin H), 100mg VITMAIN B1,100mg Lin Suanna Vitamin B2 Sodium Phosphate, 100mg vitamin B6,100mg nicotinic acid and 100mg para-amino benzoic acid, with distilled water, dissolve and be settled to 100ml.Annotate: vitamin mixture need keep in Dark Place.
The MAG solid plate: on the basis of MAG liquid nutrient medium, every liter is added 15g agar.
The structure of embodiment 1, two mutants which hads
One, the ultraviolet mutagenesis of Aspergillus nidulans wild strain A28
The spore of Aspergillus nidulans wild strain A28 is applied on the MAG solid plate that contains 50 μ g/ml HSAF to (100J/m under ultraviolet lamp
2, lethality rate is 90%) and carry out mutagenic treatment.
Two, screening and the evaluation of Aspergillus nidulans barA gene mutation body and gcs1 gene mutation body
1, the flat board after mutagenic treatment is placed in to 28 ℃ and cultivates 3 days, the bacterium colony that then picking is large is further confirmed on the MAG solid plate that contains 50 μ g/mlHSAF.
2, take mutant gene group DNA is template, and pcr amplification barA gene (ceramide synthase gene) also carries out sequencing analysis.The mutant gene group DNA of take is template, and pcr amplification gcs1 gene (ceramide glucoside transferase gene) also carries out sequencing analysis.If the barA gene is undergone mutation, the gcs1 gene is undergone mutation or barA gene and gcs1 gene are all undergone mutation, thereby cause the synthetic forfeiture of ceramide glycolipid or obviously reduce, and can be had complementary functions by corresponding wild gene, this mutant is barA gene function deletion mutant, gcs1 gene function deletion mutant or barA gene and gcs1 gene function disappearance double-mutant.
Pcr amplification barA gene the primer is to as follows:
barAF1：5’-ATCTCCGAGCTTTTATCCCAC-3’；
barAR1：5’-ATTTTCAGAACACGACATTTAGG-3’。
The annealing temperature of this primer pair is 56 ℃, and target sequence is 1639bp.
Pcr amplification gcs1 gene primer pair used is as follows:
gcs1F1：5’-GTCTCCTCGTCTTCTCTCTTCTC-3’；
gcs1R1：5’-TATTCTTTTTTTGCTTGGTTTGT-3’。
The annealing temperature of this primer pair is 56 ℃, and target sequence is 1930bp.
Two mutant have been obtained.By sequencing analysis, find: deletion mutantion has occurred in the barA gene of a mutant, causes the disappearance of 212 amino-acid residues in the middle of BarA albumen and loss of function; Nonsense mutation has occurred in the gcs1 gene of another mutant, causes producing length and be only the albumen of brachymemma of the afunction of 170 amino-acid residues.
Three, the preservation of Aspergillus nidulans barA mutant and gcs1 mutant
Aspergillus nidulans (Aspergillus nidulans) UV-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 27th, 2012 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5806.Aspergillus nidulans (Aspergillus nidulans) UV-1 claim again Aspergillus nidulans barA gene mutation body.
Aspergillus nidulans (Aspergillus nidulans) UV-13 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 27th, 2012 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5807.Aspergillus nidulans (Aspergillus nidulans) UV-13 claim again Aspergillus nidulans gcs1 gene mutation body.
The preparation of embodiment 2, antifungal factor (HSAF)
Antifungal factor (HSAF): the antifungal substance of a kind of next molten bacillus of self-produced enzyme (Lysobacter enzymogenes) C3, target is ceramide glycolipid (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).
Prepare HSAF (Yu F according to the method in document, 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, the HSAF restraining effect to wild strain and mutant strain
One, HSAF processes and morphologic observation
Aspergillus nidulans barA gene mutation body, respectively at (the first solid plate is the MAG solid plate, and the second solid plate is the MAG solid plate containing 50 μ g/ml HSAF) in the middle of two solid plates, is cultivated 3 days for 28 ℃.
Aspergillus nidulans gcs1 gene mutation body, respectively at (the first solid plate is the MAG solid plate, and the second solid plate is the MAG solid plate containing 50 μ g/ml HSAF) in the middle of two solid plates, is cultivated 3 days for 28 ℃.
Aspergillus nidulans wild strain A28, respectively at (the first solid plate is the MAG solid plate, and the second solid plate is the MAG solid plate containing 50 μ g/ml HSAF) in the middle of two solid plates, is cultivated 3 days for 28 ℃.
The results are shown in Figure 1 and Fig. 2.On the first solid plate, the growing state of three kinds of bacterial strains does not have significant difference.On the second solid plate, the growth of Aspergillus nidulans wild strain A28 is significantly inhibited (can only observe very little bacterium colony), the bacterium colony of barA gene mutation body and gcs1 gene mutation body significantly is greater than Aspergillus nidulans wild strain A28, and namely downtrod degree is less.Result shows, contains the antifungal substance that target is the ceramide glycolipid in HSAF.
Two, result verification
The spore of Aspergillus nidulans wild strain A28 is inoculated in respectively in two kinds of liquid nutrient mediums, (the first liquid nutrient medium is liquid MAG substratum, the second liquid nutrient medium is the liquid MAG substratum containing 50 μ g/mlHSAF), cultivate 8-12h, then examine under a microscope the sprouting of spore and the growth characteristics of mycelia for 28 ℃.
The results are shown in Figure 3.In the first liquid nutrient medium, spore shape is normal.In the second liquid nutrient medium, the excessive bifurcated of hypha,hyphae.This result can further confirm in HSAF to contain the antifungal substance that target is the ceramide glycolipid.
Also can be verified as follows, by the spore mycelium inoculation of Aspergillus nidulans wild strain A28, in two kinds of liquid nutrient mediums, (the first liquid nutrient medium is liquid MAG substratum, the second liquid nutrient medium is the liquid MAG substratum containing 50 μ g/mlHSAF), cultivate 3-6h for 28 ℃, then examine under a microscope the sprouting of spore and the growth characteristics of mycelia, if the excessive bifurcated of hypha,hyphae, can further confirm to contain the antifungal substance that target is the ceramide glycolipid in screened thing to be checked.
Embodiment 4, derive from the screening method of the antifungal substance that the target of microorganism is the ceramide glycolipid
One, concrete screening method
1, microbe-derived material to be checked is slightly carried, obtained material crude extract to be checked (be about to the microorganism fermentation and collect the fermentation supernatant).
2, packet transaction
Aspergillus nidulans barA gene mutation body, respectively at (the first solid plate is the MAG solid plate, and the second solid plate is the MAG solid plate containing crude extract) in the middle of two solid plates, is cultivated 3 days for 28 ℃.
Aspergillus nidulans gcs1 gene mutation body, respectively at (the first solid plate is the MAG solid plate, and the second solid plate is the MAG solid plate containing crude extract) in the middle of two solid plates, is cultivated 3 days for 28 ℃.
Aspergillus nidulans wild strain A28, respectively at (the first solid plate is the MAG solid plate, and the second solid plate is the MAG solid plate containing crude extract) in the middle of two solid plates, is cultivated 3 days for 28 ℃.
On the MAG solid plate that contains crude extract, if the mutant bacterium colony is large, its growth is not subject to the impact of this material crude extract to be checked or affects less, and the wild strain bacterium colony is less, growth is suppressed, can substantially judges in this crude extract and contain the antifungal substance that target is the ceramide glycolipid.
Two, the selection result
According to aforesaid method, the analysis of the inventor to the soil microorganisms of strain more than 900 secondary metabolite, the target that newly screens 3 its secondary metabolites of strain microorganism is the ceramide glycolipid.The active substance HB-2 that wherein representational two streptomycetes produce and HB-3 are shown in Fig. 4 to the growth effect of wild type strain A28 and Aspergillus nidulans barA gene mutation body, and active substance HB-2 and HB-3 are shown in Fig. 5 to the growth effect of wild type strain A28 and Aspergillus nidulans gcs1 gene mutation body.
Three, the further checking of the selection result
In order further filtered out microbial secondary meta-bolites to be verified, at first the spore of Aspergillus nidulans wild strain A28 or mycelium inoculation are cultivated to 8-12h or 3-6h respectively at 28 ℃ in the liquid MAG substratum that contains material crude extract to be checked, then examine under a microscope the sprouting of spore and the growth characteristics of mycelia.The microscopy result shows that 3 its secondary metabolites of strain microorganism that screen all can cause the excessive bifurcated of Aspergillus nidulans mycelia.This secondary metabolite of further having confirmed the 3 strain microorganisms that screen contains the antifungal substance that target is the ceramide glycolipid.
Take Hela cell, mouse, wheat seed and seedling etc. as material carries out biometric analysis, prove this 3 strain microbial secondary meta-bolites to the equal nontoxicity of animals and plants.
Claims (7)
1. the method for the antifungal substance that a Screening target is the ceramide glycolipid, comprise the steps A: wild fungus bacterial strain and mutant strain are cultivated containing on the solid plate of test substance respectively, if the growing state of mutant strain on flat board is better than the growing state of wild fungus bacterial strain on flat board, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance; The target of described antifungal substance is the ceramide glycolipid; Described mutant strain is for to carry out by the ceramide glycolipid metabolism approach genes involved of described wild fungus bacterial strain the bacterial strain that afunction obtains;
Ceramide glucoside transferase gene shown in the sequence 2 of the ceramide synthase gene shown in the sequence 1 that described ceramide glycolipid metabolism approach genes involved is sequence table and/or sequence table;
Described wild fungus bacterial strain is Aspergillus nidulans (Aspergillus nidulans) wild strain A28; Described mutant strain is Aspergillus nidulans (Aspergillus nidulans) UV-13 that deposit number Aspergillus nidulans (Aspergillus nidulans) UV-1 that is CGMCC No.5806 or deposit number are CGMCC No.5807.
2. the method for claim 1, it is characterized in that: described method also comprises the steps B and/or step C:
Step B: the spore of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if in the mycelia branch containing in the liquid nutrient medium of described test substance more than not containing the mycelia branch in the liquid nutrient medium of described test substance, the antifungal substance that described test substance be the candidate or candidate's the material that contains antifungal substance;
Step C: the mycelia of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if in the mycelia branch containing in the liquid nutrient medium of described test substance more than not containing the mycelia branch in the liquid nutrient medium of described test substance, the antifungal substance that described test substance be the candidate or candidate's the material that contains antifungal substance.
3. identify that whether test substance is the method for the target antifungal substance that is the ceramide glycolipid for one kind, comprise the steps D: wild fungus bacterial strain and mutant strain are cultivated containing on the solid plate of test substance respectively, if the growing state of mutant strain on flat board is better than the growing state of wild fungus bacterial strain on flat board, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance, if growing state and the wild fungus bacterial strain growing state on flat board of mutant strain on flat board do not have significant difference, the non-antifungal substance that described test substance is the candidate or candidate not containing the material of antifungal substance, the target of described antifungal substance is the ceramide glycolipid, described mutant strain is for to carry out by the ceramide glycolipid metabolism approach genes involved of described wild fungus bacterial strain the bacterial strain that afunction obtains,
Ceramide glucoside transferase gene shown in the sequence 2 of the ceramide synthase gene shown in the sequence 1 that described ceramide glycolipid metabolism approach genes involved is sequence table and/or sequence table;
Described wild fungus bacterial strain is Aspergillus nidulans (Aspergillus nidulans) wild strain A28; Described mutant strain is Aspergillus nidulans (Aspergillus nidulans) UV-13 that deposit number Aspergillus nidulans (Aspergillus nidulans) UV-1 that is CGMCC No.5806 or deposit number are CGMCC No.5807.
4. method as claimed in claim 3, it is characterized in that: described method also comprises the steps E and/or step F:
Step e: the spore of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if the mycelia branch in containing the liquid nutrient medium of described test substance is more than the mycelia branch in not containing the liquid nutrient medium of described test substance, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance, if there is no significant difference in the mycelia branch containing in the liquid nutrient medium of described test substance with the mycelia numbers of branches in not containing the liquid nutrient medium of described test substance, the non-antifungal substance that described test substance is the candidate or candidate not containing the material of antifungal substance,
Step F: the mycelia of described wild fungus bacterial strain is cultivated at the liquid nutrient medium containing described test substance or in not containing the liquid nutrient medium of described test substance respectively, if the mycelia branch in containing the liquid nutrient medium of described test substance is more than the mycelia branch in not containing the liquid nutrient medium of described test substance, the antifungal substance that described test substance is the candidate or candidate's the material containing antifungal substance, if there is no significant difference in the mycelia branch containing in the liquid nutrient medium of described test substance with the mycelia numbers of branches in not containing the liquid nutrient medium of described test substance, the non-antifungal substance that described test substance is the candidate or candidate not containing the material of antifungal substance.
5. Aspergillus nidulans (Aspergillus nidulans) UV-1, its deposit number is CGMCC No.5806.
6. Aspergillus nidulans (Aspergillus nidulans) UV-13, its deposit number is CGMCC No.5807.
7. the application of Aspergillus nidulans (Aspergillus nidulans) UV-13 that the Aspergillus nidulans that deposit number is CGMCC No.5806 (Aspergillus nidulans) UV-1 and/or deposit number are CGMCC No.5807 in the screening antifungal substance; The target of described antifungal substance is the ceramide glycolipid.
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2012
- 2012-02-28 CN CN201210048265.XA patent/CN102586394B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Interactions of antifungal plant defensins with fungal membrane components;Karin Thevissen et al;《Peptides》;20031231;第24卷;1705-1712 * |
| Karin Thevissen et al.Interactions of antifungal plant defensins with fungal membrane components.《Peptides》.2003,第24卷1705-1712. |
| 刘圣红.AbA与灰葡萄孢菌IPC合成酶相互作用的功能域及灰葡萄孢菌对植物致病生化机理的研究.《中国优秀硕士学位论文全文数据库（农业科技辑）》.2010,(第01期),D046-5. * |
| 张宏等.植物防御素研究进展.《西北师范大学学报(自然科学版)》.2006,第42卷(第5期),112-117. * |
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| CN102586394A (en) | 2012-07-18 |
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