CN117106043A - New target of albendazole in banana vascular wilt and application of albendazole in banana vascular wilt resistance - Google Patents
New target of albendazole in banana vascular wilt and application of albendazole in banana vascular wilt resistance Download PDFInfo
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- CN117106043A CN117106043A CN202310837434.6A CN202310837434A CN117106043A CN 117106043 A CN117106043 A CN 117106043A CN 202310837434 A CN202310837434 A CN 202310837434A CN 117106043 A CN117106043 A CN 117106043A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
- A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
- A01N47/18—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof containing a —O—CO—N< group, or a thio analogue thereof, directly attached to a heterocyclic or cycloaliphatic ring
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
- C12Y206/01021—D-Amino-acid transaminase (2.6.1.21), i.e. D-alanine aminotransferase/transaminase or D-aspartic aminotransferase/transaminase
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- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the technical field of biology, discloses a new target of albendazole on banana vascular wilt and application thereof in resisting banana vascular wilt, and particularly discloses a drug action target for preventing and treating banana vascular wilt, wherein the target comprises beta-tubulin and aspartate aminotransferase. According to the invention, a target spot of albendazole on banana wilt is obtained by using a drug affinity reaction target spot stabilization technology DARTS and combining with a differential gene KEGG analysis of a drug-treated thallus and a knock-out sensitivity experiment, so that the beta-tubulin and aspartate aminotransferase can be used as medicinal targets for preventing and treating banana wilt bacteria.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a novel target spot of albendazole on banana vascular wilt and application of albendazole in banana vascular wilt resistance.
Background
The fusarium oxysporum dedicated fungus, tropical race 4, race Foc TR4, is the most devastating disease in banana production worldwide, and has now spread to more than 20 regions in asia, africa, south america, ocean due to lack of effective detection techniques and measures since the outbreak in taiwan of china in 1989, resulting in a huge economic loss, called "banana cancer". Foc TR4 is soil-borne fungi, is an international plant quarantine object, and can invade banana plant vascular bundles from root systems to cause the death of the whole plant and the damage of a banana garden; the pathogen has extremely strong stress resistance and can survive in soil for 50 years, so once infected, bananas can not be planted any more within 50 years; the mutation speed of bacteria is high, 4 physiological micro-species or 24 nutrition affinity groups have evolved, and particularly, scientists are worried about a mutant strain-Tropical 4 physiological micro-species Foc TR4 which has strong pathogenicity on a world main cultivated variety-canna.
Banana wilt prevention and control is a worldwide problem, and the lack of effective prevention and control measures becomes the most main bottleneck for restricting the sustainable development of the industry. The development of the industry mainly depends on disease-resistant varieties, the quantity is small, the genotype is single, the genetic basis is narrow, and the threat of TR4 evolution is faced; biological control strains have unstable control effects due to weak colonization in soil or hosts; although technical systems such as paddy-upland rotation, rotation and interplanting of Chinese chives and bananas are established, economic and agronomic operation efficiency or benefit also need to be improved; although the chemical control has the advantages of good effect, convenient use, economy and the like, the chemical bactericide capable of effectively inhibiting the commercialization of TR4 is less, and if the chemical bactericide is used for controlling in fields and even disinfecting soil in a large amount, the chemical bactericide has serious damage to the environment, so that the search or excavation of the bactericide which is safe to the environment and human bodies, low in toxicity and high in efficiency is one of the important research contents in the future of banana wilt.
Albendazole (Albendazole) is also called enteroworm-clearing and Albendazole, and is a broad-spectrum, high-efficiency and low-toxicity benzimidazole-type human and animal insect repellent marketed by Smith Kline company in the United states in 1977. About 20 years ago, it has been a complete chemical synthesis process in China. The chemically synthesized albendazole of 13 days in 2/2012 has been registered as an agricultural bactericide as a main ingredient in chinese planting industry management department. Recently, scientists have also studied it as a specific drug for treating tumors.
Research on the mechanism of action of albendazole has focused on antiparasitic aspects. Research shows that the medicine is one kind of microtubule inhibitor and can combine with beta-microtubule protein of helminth, hookworm and other parasites to depolymerize microtubules; while inhibiting glucose uptake and transport, ultimately leading to death by parasite glycogen deficiency. In the anti-tumor research, albendazole can induce oxidative stress, promote DNA fragmentation and inhibit tumor growth; can also inhibit polymerization of tubulin, destroy microtubule function, and thereby induce tumor cell death and apoptosis. Benzimidazole drugs are used as agricultural fungus bactericides, and the mechanism research is focused on the bactericide carbendazim with broad-spectrum resistance. Carbendazim is mainly combined with beta-tubulin of fungi such as fusarium and the like, so that the carbendazim and alpha-tubulin cannot be polymerized to cause bacterial death, but the carbendazim is a single-site bactericide and extremely easy to generate drug resistance. Banana vascular wilt is reported to be naturally resistant to single-site bactericides, none of which kills spores entirely, and surviving spores eventually still lead to banana morbidity. The prior art researches find that the beta-tubulin of the carbendazim-resistant strain has reduced binding capacity with isocitrate dehydrogenase (IDH 3), thereby reducing IDH3 activity, leading to accumulation of acetyl-CoA, up-regulating DON toxin biosynthesis and increasing the content thereof. While the research on the bacteriostasis mechanism of albendazole serving as a fungus bactericide is still blank.
Disclosure of Invention
The invention aims at providing a drug action target point for preventing and treating banana fusarium wilt.
The object of the second aspect of the invention is to provide the use of beta-tubulin and aspartate aminotransferase as drug action targets in agents for controlling banana fusarium wilt.
The object of the third aspect of the invention is to provide the use of beta-tubulin and aspartate aminotransferase as drug action targets against banana vascular wilt.
The object of the fourth aspect of the present invention is to provide the use of a reagent which acts simultaneously with β -tubulin and aspartate aminotransferase for the preparation of a product for controlling banana vascular wilt.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a target for the action of a drug for controlling banana vascular wilt, said target comprising beta-tubulin and aspartate aminotransferase.
Preferably, the banana vascular wilt includes Foc TR4.
Preferably, the aspartate aminotransferase is aspartate aminotransferase in the metabolic pathway of a tricarboxylic acid cycle.
Preferably, the medicament comprises a benzimidazole compound.
Preferably, the drug comprises albendazole.
In a second aspect, the invention provides the application of beta-tubulin and aspartate aminotransferase as drug action targets in screening agents for preventing and treating banana fusarium wilt.
Preferably, the banana vascular wilt includes Foc TR4.
Preferably, the aspartate aminotransferase is aspartate aminotransferase in the metabolic pathway of a tricarboxylic acid cycle.
Preferably, the drug interacts with HIS203, TYR239, ALA238, TYR277, ALA271 and LYS272 of the aspartate aminotransferase.
Preferably, the agent has an alkylating interaction with HIS203, TYR239, ALA238, TYR277, ALA271 and a pi-alkyl interaction with LYS272 and pi-action interaction.
In a third aspect, the invention provides the use of beta-tubulin and aspartate aminotransferase as drug action targets in combating banana vascular wilt.
Preferably, the banana vascular wilt includes Foc TR4.
Preferably, the aspartate aminotransferase is aspartate aminotransferase in the metabolic pathway of a tricarboxylic acid cycle.
Preferably, the drug interacts with HIS203, TYR239, ALA238, TYR277, ALA271 and LYS272 of the aspartate aminotransferase.
Preferably, the agent has an alkylating interaction with HIS203, TYR239, ALA238, TYR277, ALA271 and a pi-alkyl interaction with LYS272 and pi-action interaction.
In a third aspect, the invention provides the use of an agent which acts simultaneously with β -tubulin and aspartate aminotransferase for the preparation of a product for controlling banana vascular wilt.
Preferably, the reagent comprises a benzimidazole compound.
Preferably, the agent comprises albendazole.
Preferably, the product comprises a biocide.
The beneficial effects of the invention are as follows:
the invention utilizes a drug affinity reaction target stabilization technology DARTS, combines differential gene KEGG expression analysis of drug-treated thalli and utilizes knockout sensitivity experiments to obtain the target of albendazole on banana vascular wilt, wherein the target comprises beta-tubulin and aspartate aminotransferase. Further through homologous modeling and molecular docking, the binding energy of the aspartate aminotransferase and the albendazole is 6.4kcal/mol, the albendazole has alkylation interaction and pi-alkyl interaction with HIS203, TYR239, ALA238, TYR277 and ALA271, and pi-action interaction with LYS272, and the aspartate aminotransferase is suggested to be used as a new target spot of banana fusarium wilt bacteria in the control of banana fusarium wilt bacteria.
Drawings
FIG. 1 is a graph of the effect of various concentrations of albendazole on spore germination rates of Foc TR4.
FIG. 2 is a graph showing the effect of various concentrations of albendazole on the sporulation rate of Foc TR4.
FIG. 3 is a graph showing the effect of albendazole on Foc TR4 hypha superoxide dismutase content.
FIG. 4 is a projection electron microscope image of albendazole treated Foc TR4 hyphae.
FIG. 5 is a graph showing the results of differential gene KEGG analysis.
FIG. 6 is a graph showing the results of DARTS experimental polyacrylamide gel electrophoresis.
FIG. 7 is a graph showing the results of susceptibility of tubulin knockouts and candidate target protein knockouts to albendazole.
FIG. 8 is a graph showing the molecular docking results of aspartate aminotransferase with albendazole.
Detailed Description
The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.
The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.
Example 1
The example is used for examining the influence of albendazole on the spore yield and spore germination rate of Foc TR4, and specifically comprises the following steps:
measurement of spore production: fresh spore suspension of Foc TR4 strain (1 mL, 1X 10 5 CFU/mL spores) were inoculated into 50mL of PDB medium, respectively, and a solution of albendazole diluted with DMSO in a sterile environment (final concentration of albendazole in the culture system was 1.7. Mu.g/mL and 3.4. Mu.g/mL) was added, while DMSO was added in the same ratio as the control group. Shaking and mixing uniformly, and culturing for 2d in dark shaking at 28 ℃ and 180 r/min. After the cultivation, the mycelia were filtered with 5 layers of lens paper, spores were collected in 50mL centrifuge tubes, centrifuged at 6000r/min for 10min, the supernatant was decanted, diluted to 1mL with sterile water, the number of conidia was counted with a hemocytometer, and each treatment was repeated 6 times.
Determination of spore germination rate: fresh spore suspension of Foc TR4 strain (1 mL, 1X 10 5 CFU/mL spores) were inoculated into 50mL of PDB medium, respectively, and a solution of albendazole diluted with DMSO in a sterile environment (final concentration of albendazole in the culture system was 1.7. Mu.g/mL and 3.4. Mu.g/mL) was added, while DMSO was added in the same ratio as the control group. Shaking and mixing uniformly, and culturing in dark at 28 ℃ and 180r/min for 18h. Sucking spore suspension drop onto glass slide after culturing, covering with cover glass, and counting spore germination rate under 20 times mirror, wherein spore germination is obtained by spore bud tube length greater than half spore lengthEach treatment was repeated 3 times. Spore germination rate was calculated according to the following calculation formula "= number of germinated steamed stuffed bun/total number of statistical spores x 100%.
By treating spores of Foc TR4 strain with albendazole solutions of different concentrations, the results showed that the sporulation rate and spore germination rate of Foc TR4 gradually decreased as albendazole concentration increased. When the concentration of albendazole was 1.7. Mu.g/mL, the spore germination rate was reduced to about 50% (FIG. 1), and the spore yield was reduced by 1/3 (FIG. 2).
Example 2
The example is used for examining the influence of albendazole on the content of superoxide dismutase (SOD) in Foc TR4 hyphae, and is specifically as follows:
fresh spore suspension of Foc TR4 strain (1 mL, 1X 10 5 CFU/mL spores) were inoculated into 50mL of PDB medium, respectively, and a solution of albendazole diluted with DMSO in a sterile environment (final concentration of albendazole in the culture system was 1.7. Mu.g/mL and 3.4. Mu.g/mL) was added, while DMSO was added in the same ratio as the control group. Shaking and mixing uniformly, and culturing for 5 days in dark shaking at 28 ℃ at 180 r/min. After the completion of the cultivation, mycelia were filtered with 5 layers of mirror paper and collected, and the collected mycelia were assayed using a superoxide dismutase kit (D799594-0100) from Shanghai Co., ltd, and each treatment was repeated 8 times.
As shown in FIG. 3, the content of SOD in the mycelia was significantly reduced by treating spores of Foc TR4 strain with albendazole solution, which had no significant effect on the content of SOD in the mycelia, compared to the DMSO-treated mycelia (60.3U/g).
Example 3
The example was used to examine the effect of albendazole on the form of Foc TR4 spores, as follows:
taking wild type Foc TR4 bacterial blocks and bacterial blocks treated by 1.7mg/L and 3.4mg/L albendazole solution (diluted by DMSO) respectively, placing the bacterial blocks in 2.5% glutaraldehyde solution for fixation overnight, then fixing the bacterial blocks in 1% osmium acid solution for 4 hours, dehydrating the bacterial blocks by concentration gradient ethanol (30% -50% -70% -80% -90%), treating the bacterial blocks by absolute ethanol for 20 minutes, and treating the bacterial blocks by acetone for 20 minutes. And then treating the sample by using a mixed solution of an embedding agent and acetone (V/V=3/1), embedding the treated sample, slicing the sample in an ultrathin slicing machine, and finally observing and photographing by using a transmission electron microscope.
The results of observation of spore germination status by microscope using growth status of wild strain as control show that albendazole invaginates spore plasma membrane, and degradation of organelle occurs with increase of drug concentration, spore cavitation (figure 4), revealing influence of albendazole on Foc TR4 spore morphology.
Example 4
This example was used to examine the potential target of albendazole in Foc TR4.
The effect of albendazole on Foc TR4 transcript levels was studied by RNA-seq, specifically: RNA of wild type, 1.7mg/L and 3.4mg/L albendazole treated strains were extracted, respectively, the Hua megagene was sent to perform RNA-seq analysis, and the differential gene, which plays an important role in the pathogenic process, of 1.7mg/L and 3.4mg/L albendazole treated strains compared with the wild type was subjected to KEGG metabolic pathway analysis, and at least 3 biological repeats were performed. KEGG-related analysis showed that the differential genes are mainly enriched in the amino acid biosynthetic pathway, the 2-oxo carboxylic acid metabolic pathway, and the three metabolic pathways of glyoxylic acid and dicarboxylic acid metabolism, in addition to the secondary metabolic-related pathways (fig. 5). Through KEGG pathway patterns, it was found that tricarboxylic acid metabolism (TCA) -aspartic acid occurs in all three metabolic processes, with up-regulated genes enriched in the aspartate-oxaloacetate-citrate pathway; whereas downregulation of the gene enriches the aspartate-L-4-aspartate phosphate pathway (H), suggesting that tricarboxylic acid metabolism (TCA) -aspartate aminotransferase may be a potential target for albendazole for Foc TR4.
Meanwhile, the Foc TR4 total protein with the same concentration and the albendazole water saturated solution are incubated for 2 hours at 37 ℃, and DMSO is used as a blank control. After incubation, add 1:200 and 1 in addition to the blank: 300, and performing enzymolysis on the enzymolysis liquid at room temperature for 45min. The enzymolysis sample is separated by polyacrylamide gel electrophoresis (SDS-PAGE), compared with a blank control, the stable undegraded band is regarded as a candidate protein band, and the candidate protein band is cut and stored for mass spectrum identification and compared with a database to identify candidate binding proteins. The results show that distinct bands can be seen by SDS-PAGE (FIG. 6, indicated by arrows), and that 2 genes associated with TCA metabolic pathways were selected as candidate target proteins (pyruvate carboxylase and aspartate aminotransferase) based on subsequent mass spectrometry binding to RNA-seq.
Further verifying sensitivity of benzimidazole universal target tubulin knockouts and candidate target protein knockouts to albendazole by using a hypha growth rate method, namely detecting whether Foc TR4 knockouts can grow on an albendazole flat plate containing 10ppm by using the hypha growth rate method, specifically comprising the following steps: placing the constructed candidate target protein knockouts (containing the knockouts of benzimidazole universal target beta-tubulin) on an albendazole flat plate containing 10ppm, observing the growth state of hyphae, and reducing the sensitivity to albendazole to obtain the target protein knockouts. The construction method of the Foc TR4 knockout is as follows: the target protein deletion mutant strains, namely Foc TR4 knockdown strains (respectively designated as Delta 05441, delta15265, delta 05875, delta 01180, delta 01510, delta03170 and Delta 07863) were constructed by constructing DNA fusion fragments comprising tubulin (FOIG_ 05441, FOIG_15265, FOIG_05875, FOIG_01180 and FOIG_ 01510), pyruvate carboxylase (FOIG_03170), aspartate aminotransferase (FOIG_ 07863) and the like, respectively, upstream of the target gene, hygromycin gene (HPH), and downstream of the target gene by a fusion PCR (double-joint PCR) method, and transferring vectors containing the DNA fusion fragments into protoplasts prepared by Foc TR4 by using a PEG-mediated method.
Experiments have found that three alpha tubulin (Δ 05441, Δ 01180 and Δ 01510) and pyruvate carboxylase (Δ03170) are sensitive to albendazole; two beta tubulin (delta 15265, delta 05875) have reduced sensitivity to albendazole, but the reduction degree is different, wherein the reduction degree of delta 05875 to albendazole is larger, which indicates that the beta tubulin is a direct target of albendazole; the decreased sensitivity of aspartate aminotransferase (Δ 07863) to albendazole suggests that aspartate aminotransferase is a target for albendazole (fig. 7). Analysis of the Foc TR4 genome revealed that aspartate aminotransferase was a single copy gene in the genome.
Further uploading an aspartate aminotransferase amino acid sequence file to a Swiss Model server, searching protein crystal data with high fitness through sequence alignment, removing protein crystal water, original ligand and the like by using Pymol2.3.0, and establishing a protein skeleton. Protein binding sites were predicted using POCAASA 1.1, docking was performed with AutoDock Vina1.1.2, aspartate aminotransferase related parameters were set as: center_x= -0.6, center_y= -2.7, center_z= -3.3; search space: size_x:60, size_y:60, size_z:60 (pitch of each lattice is) 10, and the rest parameters are default settings.
The result of molecular docking of aspartate aminotransferase and albendazole shows that the binding energy of the aspartate aminotransferase and albendazole is 6.4kcal/mol, and the aspartate aminotransferase and albendazole have better binding effect. Albendazole has alkylation interactions with HIS203, TYR239, ALA238, TYR277, ALA271 and pi-alkyl interactions with LYS272 (fig. 8).
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A target for the action of a drug against banana vascular wilt, said target comprising β -tubulin and aspartate aminotransferase.
2. The action target according to claim 1, wherein the banana vascular wilt comprises Foc TR4.
3. The action target according to claim 2, wherein the aspartate aminotransferase is aspartate aminotransferase in the tricarboxylic acid cycle metabolic pathway.
4. The action target according to claim 3, wherein the drug comprises a benzimidazole compound.
5. The application of beta-tubulin and aspartate aminotransferase as drug action targets in screening agents for preventing and treating banana fusarium wilt.
6. The application of beta-tubulin and aspartate aminotransferase as drug action targets in resisting banana fusarium wilt.
7. The use according to claim 5 or 6, wherein the banana vascular wilt comprises Foc TR4.
8. The use according to claim 7, wherein the medicament interacts with HIS203, TYR239, ALA238, TYR277, ALA271 and LYS272 of the aspartate aminotransferase.
9. The use according to claim 8, characterized in that the aspartate aminotransferase is aspartate aminotransferase in the tricarboxylic acid cycle metabolic pathway.
10. The application of the reagent which simultaneously acts with beta-tubulin and aspartate aminotransferase in preparing the product for preventing and treating banana vascular wilt.
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