Drug target protein CtSR

Abstract

The invention relates to a drug target protein CtSR, which comprises a wild type strain, a knockout mutant delta CtSR and a supplementary strain CtSR-C. The knockout mutant delta CtSR has been preserved and is named as CtRR13, the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, the preservation date is March 22, 2024, the preservation number is CGMCC No.41274, and the preservation address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. After the technology is adopted, the gene has the following advantages that the pathogenicity of the gene is greatly reduced due to deletion of the gene, and the sensitivity of pathogenic bacteria to triazole medicaments is greatly improved due to deletion of the gene, so that the gene can be used as a targeted selective medicament or a material for preparing and screening medicaments, and has a safe and efficient application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural biological medicines, and in particular to a drug target protein CtSR. Background Art

[0002] The pathogen of anthracnose, Colletotrichum spp., can infect a variety of important fruits, vegetables, cereals, grasses, and ornamental plants. C. truncatum is one of the typical anthracnose pathogens with a wide host range, with more than 460 host plant species recorded. Currently, chemical control remains the main method for controlling anthracnose, as fully resistant and commercially viable fruit or vegetable varieties have not yet been developed. Among the limited chemical control methods for anthracnose, sterol demethylation inhibitors (DMIs) stand out due to their effective protective and curative properties and have been successfully used in disease control for many years (Chen et al., 2018). However, it has been reported that Colletotrichum spp. has inherent resistance to certain DMIs, and there have been reports of failure of DMI-type agents to control anthracnose in a variety of fruits, vegetables, and field crops, including grapes, soybeans, and peppers (Deng et al., 2011; Zhang et al., 2013; Chen et al., 2022; Wei et al., 2022). Therefore, discovering new molecular targets for fungicides and developing new fungicides with selectivity and high activity are of great significance for the prevention and control of major crop diseases. Summary of the invention

[0003] The present invention aims to solve the above technical problems and provides a drug target protein CtSR, which can be used as a molecular target for drug development and provide a basis for the development of fungal disease drugs. The purpose of the present invention is also to use compounds with CtSR as a receptor in the prevention and treatment of plant fungal diseases. The present invention provides a drug target protein CtSR, and the amino acid sequence of the CtSR protein is shown in SEQ ID NO: 1. The present invention provides a gene encoding the drug target CtSR protein, and the nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0004] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0005] A drug target protein CtSR, including a wild-type strain CtRR13, a knockout mutant ΔCtSR and a complemented strain CtSR-C, wherein the knockout mutant ΔCtSR is classified and named Colletotrichum truncatum, and the deposit unit is the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is March 22, 2024, the deposit number is CGMCC No.41274, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] A drug target protein CtSR, the amino acid sequence of the protein CtSR is shown in SEQ ID NO: 1.

[0007] The gene encoding the drug target CtSR protein, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0008] Application of a compound with CtSR protein as receptor or a pharmaceutically acceptable salt thereof in preventing and controlling plant fungal diseases.

[0009] The fungal diseases include plant diseases caused by anthracnose.

[0010] Plant diseases caused by anthracnose include soybean anthracnose and vegetable and fruit anthracnose.

[0011] Including wild-type strains, knockout mutants ΔCtSR and complemented strains CtSR-C. The knockout mutant ΔCtSR has been deposited at the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is March 22, 2024, the deposit number is CGMCC No.41274, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] The application of the drug target CtSR protein mutant in screening specific agents for controlling plant fungal pathogens and in reducing the pathogenicity of plant fungal pathogens,

[0013] After adopting the above technology, the present invention has the following advantages:

[0014] The present invention finds that the deletion of this gene will greatly reduce its pathogenicity, and the deletion of this gene will lead to a significant increase in the sensitivity of pathogens to triazole drugs. Therefore, it can be used as a targeted selective drug or for preparing and screening drug materials, and has a safe and efficient application prospect.

[0015] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or technical descriptions will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 is a phylogenetic analysis diagram of the transcription factor CtSR of the present invention;

[0018] Figure 2 It is the construction of the transcription factor CtSR complementation strain of the present invention and the diagram of the triazole drugs;

[0019] Figure 3 It is a graph showing the sensitivity changes of the ΔCtSR of the present invention, the revertant and its parent wild strain to different types of fungicides;

[0020] Figure 4 This is a schematic diagram of constructing a gene knockout vector using fusion PCR of the present invention;

[0021] Figure 5 This is a diagram showing the effect of knocking out CtSR on CYP51s gene expression and ergosterol synthesis;

[0022] Figure 6 It is a heat map analysis diagram of differential genes in the ergosterol synthesis pathway of the present invention;

[0023] Figure 7 is the GO classification diagram of differentially expressed genes of the present invention;

[0024] Figure 8 It is the KEGG signaling pathway diagram of the top 20 significantly enriched differentially expressed genes of the present invention;

[0025] Fig. 9 It is a graph for determining the pathogenicity of the wild-type and mutant strains of the present invention. DETAILED DESCRIPTION

[0026] Specific embodiments of the present invention will now be mentioned in detail. Although the present invention is described in conjunction with these specific embodiments, it should be appreciated that it is not intended to limit the present invention to these specific embodiments. On the contrary, these embodiments are intended to cover substitutions, changes or equivalent embodiments that may be included in the spirit and scope of the invention defined by the claims. In the following description, a large number of specific details are set forth in order to provide a comprehensive understanding of the present invention. The present invention can be implemented without some or all of these specific details. In other cases, in order not to make the present invention unnecessarily obscure, well-known process operations are not described in detail.

[0027] When used in conjunction with "including," "methods comprising," or similar language in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The present invention is further described in detail below in conjunction with the full text.

[0029] Combined with Figure 1-Figure 9 , a drug target protein CtSR, including a wild-type strain, a knockout mutant ΔCtSR and a complemented strain CtSR-C. The knockout mutant ΔCtSR has been deposited and named CtRR13. The depositor is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is March 22, 2024. The deposit number is CGMCCNo.41274. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0030] Embodiment 1:

[0031] In the present invention, in order to verify the biological function of CtSR protein, a knockout vector ΔCtSR and a recovery vector ΔCtSR-C were constructed by gene knockout method, and the specific steps are as follows:

[0032] Construction process of knockout and complementation strains:

[0033] Construction of knockout vector ΔCtSR: This study mainly uses the split marker method (SplitMarker) to construct the DNA fragment required for knockout. The specific steps are as follows:

[0034] 1) Design primers to amplify the upstream and downstream sequences of the target gene. The reverse primer for amplifying the upstream sequence and the forward primer for amplifying the downstream sequence both contain a 25 bp hygromycin gene linker sequence;

[0035] 2) The upstream and downstream fragments amplified in the first step were cut and recovered. The purified upstream and downstream fragments were then mixed with the hygromycin gene for fusion PCR. The reaction conditions were: 94°C for 2 min; 94°C for 30 s, 60°C for 10 min, 72°C for 5 min, for a total of 10 cycles; 72°C for 10 min. The reaction system was:

[0036]

[0037]

[0038] 3) The PCR product of the second step was used as a template, and primers P1 / P2 and P3 / P4 were used to amplify two DNA sequences, namely, the upstream of the target gene-hygromycin gene and the downstream of the target gene, and the PCR product was cut and recovered for protoplast transformation (such as Figure 4 shown).

[0039] 4) After the target gene is replaced by hygromycin, the knockout mutant obtained can grow normally on PDA medium containing 200μg / mL hygromycin hygB. The knockout mutant initially obtained was cultured for multiple generations on PDA medium containing 200μg / mL hygromycin hygB. After selecting the strain that can grow stably in PDA medium containing 200μg / mL hygromycin, extract its DNA and use PCR to identify whether the target gene is replaced by the hygromycin gene. A total of three pairs of primers were selected for verifying the knockout transformants. The hygromycin gene fragment was amplified by hygromycin amplification primers Hyg-F / Hyg-R.

[0040] CTRU02_gene-5-YZ-F / CTRU02_gene-5-YZ-R and

[0041] CTRU02_gene-3-YZ-F / CTRU02_gene-3-YZ-R respectively amplified the downstream junction region fragments of the target gene in the knockout mutant.

[0042] PEG-mediated preparation and transformation of C. truncatum protoplasts:

[0043] Inoculate three 5 mm bacterial cakes into 50 mL PDA medium and shake at 27°C and 200 r / min for 36 h.

[0044] Collect the mycelium by filtering through 3 layers of sterile lens paper and rinse twice.

[0045] 10 mL of 0.7 M NaCl solution was used to dissolve 0.075 g of Lysing Enzymes from Trichodemharzimum, and then 10 mL of 0.7 M NaCl solution was used to dissolve the solution, and the dissolved solution was passed through a 0.22 μm bacterial filter.

[0046] The above enzymatic hydrolysate and mycelium were transferred into a 50 mL conical flask and lysed on a shaker at 30 °C and 80 r / min for 3 h.

[0047] The mycelial lysate in step 4 was filtered into a 50 mL centrifuge tube, and it was repeatedly rinsed with 0.7 M NaCl solution, and the filtrate was collected.

[0048] Precool the centrifuge to 4°C, place the filtrate obtained in the previous step into the centrifuge, centrifuge at 4°C, 4000r / min for 10min, and discard the supernatant.

[0049] Pipette 1 mL of STC solution to suspend the precipitate, centrifuge at 4000 rpm at 4°C for 10 min, and discard the supernatant.

[0050] Put the above suspension into a 10 mL glass test tube, then add 35 μL of purified gene fragments along the wall of the tube, gently blow and mix with a pipette, and let it stand on ice for 15 minutes.

[0051] Add 200 μL, 200 μL, and 800 μL of PEG solution along the wall of the test tube in sequence, gently rotate the test tube to mix thoroughly, and place it on ice for 15 minutes.

[0052] Slowly add 1 mL of STC solution along the wall of the test tube and gently rotate the test tube to mix thoroughly.

[0053] Take 450 μL of the mixed solution prepared above, mix it thoroughly with the regeneration medium at about 45° C. in a culture dish, and place it in an incubator at 27° C. for dark culture.

[0054] After 1 day, the newly germinated protoplasts were covered with PDA medium containing 200 μg / mL hygromycin and placed in an incubator at 27°C in the dark.

[0055] After 4 days, single colonies with good growth on the surface of the culture medium were picked and cultured on the screening medium for 4 generations.

[0056] Construction of ΔCtSR-C restoration vector: Using the genome of C. truncatum wild-type strain as a template, the CtSR promoter (about 1200 bp sequence), open reading frame (ORF) and terminator (about 500 bp sequence) were amplified with primers CtSR-CF-SacⅠ / CtSR-CF-SmaⅠ, and then connected to plasmid pCETNS-4. Plasmid pCETNS-4 contains KanR gene and NeoR gene. The restriction sites were selected as SmaⅠ and SacⅠ. The constructed vector was linearized with SacⅠ endonuclease, and the linearized plasmid was introduced into the knockout by PEG-mediated protoplast transformation. The complementing transformants were screened for resistance using G418 containing 800 μg / mL to obtain the complementing strain.

[0057] Embodiment 2:

[0058] CtSR gene is involved in regulating the sensitivity of anthrax to DMI

[0059] The homologous genes of CTRU02_06681 gene were compared through the NCBI website, and the phylogenetic analysis of the amino acid sequence of its homologous protein was performed through MEGA7 software. The results showed that the homologous protein of CTRU02_06681 only appeared in the class of dung fungi and hammer tongue fungi, and was not found in the subphylum of basidiomycetes, subphylum of exocystaceae and subphylum of yeast. The gene was named CtSR ( Figure 1 ).

[0060] To further confirm that the changes in the mutant's performance were caused by CtSR, the CtSR gene was restored to the knockout mutant. Subsequently, the restored strain was placed on a medium containing 1 μg / mL tebuconazole and 0.1 μg / mL difenoconazole (both are triazole drugs) to observe the colony morphology ( Figure 2 ). A is a schematic diagram of vector construction; B is PCR detection of complemented strains; C is the sensitivity of the complemented strains to difenoconazole and tebuconazole.

[0061] The results showed that the complemented strain CtSR-C restored its resistance to triazoles, which indicated that CtSR was involved in regulating the sensitivity of flat-headed anthracnose fungi to triazoles.

[0062] The sensitivity of the wild-type strain, the knockout mutant ΔCtSR, and the complemented strain ΔCtSR-C to other triazoles and other action site agents was further tested. The wild-type strain and the complemented strain could grow normally on PDA plates containing fludioxonil, prochloraz, diniconazole, and fluopicolide (all triazoles) without significant differences, and the growth of the knockout mutant strain on these drug-containing media was completely inhibited. Correspondingly, on PDA plates containing 0.05μg / mL fludioxonil, 0.1μg / mL pyraclostrobin, 0.1μg / mL fluazinam, and 0.1μg / mL carbendazim (other site fungicides), all three strains could grow. The EC50 values ​​of fludioxonil, pyraclostrobin, fluazinam, and carbendazim against the three strains were further determined, indicating that there was no significant difference in the sensitivity of the wild-type strain and the mutant strain to fungicides of other action types (p<0.05). Therefore, the transcription factor CtSR is only involved in regulating the sensitivity of C. truncatum to DMI agents (e.g. Figure 3 shown).

[0063] Embodiment 3:

[0064] CtSR regulates CYP51 gene expression and ergosterol biosynthesis in C. truncatum:

[0065] The targets of triazole drugs in C. truncatum are CYP51A and CYP51B genes. The expression of CYP51 genes in wild-type strains and knockout strains was measured under 2μg / mL difenoconazole treatment. The results showed that regardless of whether the drug was added or not, the expression of CYP51A and CYP51B in ΔCtSR was significantly lower than that in the wild type, and the drug treatment could no longer induce the expression of CYP51A and CYP51B in ΔCtSR ( Figure 5A). This indicates that the transcription factor CtSR can regulate the expression of the CYP51 gene. Furthermore, the ergosterol content in the wild type, knockout strain and complement strain was determined by high performance liquid chromatography. After knocking out CtSR, the ergosterol content was significantly reduced, while the ergosterol content of the complement strain ΔCtSR-C was not significantly different from that of the wild type ( Figure 5 B), indicating that CtSR regulates ergosterol biosynthesis in C. truncatum.

[0066] Embodiment 4:

[0067] Transcription factor CtSR is involved in regulating ergosterol biosynthesis pathway genes:

[0068] In order to further clarify the effect of CtSR on the gene expression of ergosterol biosynthesis pathway, differential gene comparison was performed in different comparison groups, and heat map was drawn for analysis ( Figure 6 ). The results showed that some genes in the sterol synthesis pathway were able to respond to the induction of difenoconazole in the wild-type strain, but after knocking out CtSR, the ability of these genes to respond to difenoconazole was greatly weakened, or they could not respond to drug induction, indicating that these genes may be regulated by the transcription factor CtSR. Therefore, it is speculated that the transcription factor CtSR may affect the sensitivity of C. truncatum to DMI drugs by affecting the expression of these sterol synthesis pathway genes.

[0069] like Figure 7As shown in the figure, GO analysis showed that a total of 206 genes were classified and annotated in WT and case2 (ΔCtSR) samples. These genes mainly included three categories, namely biological processes, cellular components and molecular function. Among them, biological processes included a total of 16 subcategories, and the order of differential gene enrichment was cellular process, metabolic process and localization. In cellular components, there were a total of 7 subcategories, and the order of differential gene enrichment was organelle, membrane and membrane-enclosed lumen. In molecular function, there were a total of 5 subcategories, and the order of differential gene enrichment was catalytic activity, binding activity and transporter activity. In case1 (WT + 2μg / mL difenoconazole) and case3 (ΔCtSR + 2μg / mL difenoconazole), a total of 599 genes were classified and annotated. These genes mainly include three categories, namely biological processes, cellular components and molecular function. Among them, biological processes contains a total of 17 subcategories, which are cellular process, metabolic process and localization in order of differential gene enrichment. In cellular components, there are a total of 7 subcategories, which are organelle, membrane and membrane-enclosed lumen in order of differential gene enrichment. In molecular function, there are a total of 9 subcategories, which are catalytic activity, binding activity and transporter activity in order of differential gene enrichment.Taken together, the above results indicate that the transcription factor CtSR is involved in regulating the biological processes, cellular components and molecular functions of C. truncatum.

[0070] like Figure 8 As shown in the figure, the larger the bubble in the bubble chart, the more differential protein-coding genes the pathway contains, the smaller the enrichment P value, and the greater the significance. KEGG enrichment analysis shows that the differential genes are annotated to 55 metabolic pathways in total. The differentially expressed genes of WT and case2 (ΔCtSR) samples, as well as case1 (WT + 2μg / mL difenoconazole) and case3 (ΔCtSR + 2μg / mL difenoconazole) samples are enriched in the sterol biosynthesis pathway. This indicates the potential role of the transcription factor CtSR in the sterol metabolic pathway, which happens to be the target of triazole drugs.

[0071] Embodiment 5:

[0072] Effect of CtSR on pathogenicity:

[0073] like Fig. 9 As shown, pathogenicity is an important indicator to measure the infectivity of pathogens. The effect of CtSR on pathogenicity was determined. The diameters of lesions produced by the wild-type strain WT, the knockout mutant ΔCtSR, and the complemented strain CtSR-C on peppers were 64.14 mm, 10.14 mm, and 63.71 mm, respectively. Fig. 9 ). The results showed that both the wild-type strain and the knockout mutant could infect pepper fruits, but there was a significant difference in the diameter of the lesions produced (p<0.05). Under the same conditions, the diameter of the lesions produced by the knockout mutant was significantly smaller than that of the wild-type strain. After knocking out the transcription factor CtSR, the pathogenicity of Colletotrichum flatheadensis was significantly reduced. This indicates that the transcription factor CtSR is involved in regulating the pathogenicity of C. truncatum.

[0074] The amino acid sequence of the target protein CtSR is shown in SEQ ID NO: 1:

[0075] mrrshkksragckrcksrkkcdvhrcgncvkhgvscdndddtatsnttsssrasstassagsyrtrtstgastaactyttsnaamsattsnnrrmhtsmtshtvvntathdwntvrasgasyadamavaahrsnddgarashsymasaaaycscngtsnastaaasstatrrdadndssaysswhagvktvvatswwrnsgvdsvddatstsgknmdrndaamstrsyhavavnwahkhrgaaaatvskrrrraaacakgsavwwgvsrrvmgvsatswwghwvraykhsvwgsdwvtskakgnnmsnsvnhtgvarnvvthav

[0076] The nucleotide sequence of the target protein CtSR, SEQ ID NO: 2, is as follows:

[0077]

[0078] The present invention and its embodiments are described above, and such description is not restrictive. What is shown in the full text is only one of the embodiments of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A drug target protein CtSR, characterized in that: The amino acid sequence of the protein CtSR is shown in SEQ ID NO:

1.

2. A gene encoding the drug target CtSR protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. Use of a compound using the CtSR protein according to claim 1 as a receptor or a pharmaceutically acceptable salt thereof in preventing and controlling plant fungal diseases.

4. The use according to claim 3, characterized in that: The fungal diseases include soybean anthracnose, vegetable and fruit anthracnose, wheat fusarium rust, rice seedling bakan and gray mold.

5. The drug target protein CtSR according to claim 1, characterized in that: Including wild-type strains, knockout mutants ΔCtSR and complemented strains CtSR-C. The knockout mutant ΔCtSR has been deposited at the Institute of Microbiology, Chinese Academy of Sciences. The deposit date is March 22, 2024. The deposit number is CGMCC No.41274. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

6. A drug target protein CtSR according to claim 6, characterized in that: The drug target CtSR protein mutant is used in screening specific agents for preventing and controlling plant fungal pathogens and in reducing the pathogenicity of plant fungal pathogens.

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