CcABCB5 protein and encoding gene of Corynespora polymyxinii and its application

Abstract

The present invention discloses a CcABCB5 protein and a coding gene of polymyxin Bacillus and their application. The present invention belongs to the field of biotechnology, and specifically relates to a CcABCB5 protein and a coding gene of polymyxin Bacillus and their application. The protein of the present invention is any one of the following proteins: A1) a protein whose amino acid sequence is shown in SEQ ID No.3; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1); A3) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2). The CcABCB5 protein can participate in regulating the resistance of polymyxin Bacillus to fungicides.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a polyhomocybin CcABCB5 protein and a coding gene and applications thereof. Background Art

[0002] Corynosporium polysporum is an important plant pathogen. It is the earliest discovered species in the genus Corynosporium and has the widest host range. It can infect most vegetables such as Cucurbitaceae, Solanaceae and Leguminosae, horticultural flowers, economic crops such as rubber and food crops such as soybean, about 380 genera and more than 530 species of plants. Corynosporium leaf spot of cucumber occurs on a large scale in many provinces in my country, such as Beijing, Shandong, Gansu, Hebei, Guangdong and Heilongjiang, as well as in Japan and South Korea. The general field incidence rate is 10% to 25%, and in severe cases it can reach 60% to 70%, or even 100%. Therefore, Corynosporium leaf spot of cucumber caused by Corynosporium polysporum has become an important disease on cucumber. At present, chemical agents are the most important means of preventing and controlling this disease. Chemical agents that have been reported to be effective against Corynosporium leaf spot of cucumber at home and abroad mainly include succinate dehydrogenase inhibitors (SDHIs), quinone outside inhibitors (QoIs), sterol biosynthesis inhibitors (SBIs), etc. However, as chemical agents are gradually applied to the market, resistance issues are constantly reported. ABC (ATP-binding cassette) transporters are a type of protein structure located on the cell membrane of pathogens, which can prevent the accumulation of toxic substances in pathogen cells to reach lethal drug concentrations. Overexpression of this protein leads to the generation of drug resistance. Summary of the invention

[0003] The main problem to be solved by the present invention is how to regulate the resistance of pathogens to fungicides.

[0004] In order to solve the above problems, the present invention provides a protein for regulating the resistance of pathogens to fungicides.

[0005] The protein provided by the present invention may be any of the following proteins:

[0006] A1) a protein with an amino acid sequence as shown in SEQ ID No. 3;

[0007] A2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of A1) and having 80% or more identity with the protein of A1) and having the same function;

[0008] A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] The name of the protein described in A1) above is CcABCB5.

[0010] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 3 in the sequence listing.

[0011] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0012] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0013] A person skilled in the art can easily mutate the nucleotide sequence encoding protein CcABCB5 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of protein CcABCB5 isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode protein CcABCB5 and have the function of protein CcABCB5.

[0014] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0015] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for a pair of amino acid sequences or nucleotide sequences to calculate the identity, then the value (%) of the identity can be obtained.

[0016] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0017] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0018] In the above, the protein is derived from Corynespora cassiicola.

[0019] The present invention also provides a biological material related to the above protein, and the biological material may be any one of the following:

[0020] B1) a nucleic acid molecule encoding the protein described above;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0024] C1) a nucleic acid molecule that inhibits, reduces or silences the expression of a gene encoding the protein described above;

[0025] C2) expressing the gene encoding the nucleic acid molecule described in C1);

[0026] C3) an expression cassette containing the coding gene described in C2);

[0027] C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3);

[0028] C5) A recombinant microorganism containing the coding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4).

[0029] In the above biological material, the nucleic acid molecule described in B1) is the gene shown in E1) or E2) below:

[0030] E1) the coding sequence is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

[0031] E2) The nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No.1.

[0032] The DNA molecule shown in SEQ ID No. 2 (CcABCB5 gene regulating resistance of pathogenic bacteria to fungicides) encodes a protein CcABCB5 having an amino acid sequence of SEQ ID No. 3.

[0033] The nucleotide sequence shown in SEQ ID No. 2 is the nucleotide sequence of the gene (CDS) encoding protein CcABCB5.

[0034] The CcABCB5 gene of the present invention can be any nucleotide sequence capable of encoding protein CcABCB5. Considering the degeneracy of codons and the preference of codons of different species, those skilled in the art can use codons suitable for expression of a specific species as needed.

[0035] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by modifying the codon preference based on the nucleotide sequence shown in SEQ ID No. 2.

[0036] B1) The nucleic acid molecule may also include a nucleic acid molecule having a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No. 2 and originating from the same species.

[0037] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0038] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, the vectors may be pPIC9K, pPIC3.5, etc.

[0039] By using any vector that can guide the expression of foreign genes in microorganisms, the CcABCB5 gene or gene fragment provided by the present invention is introduced into microbial cells or recipient microorganisms to obtain transgenic microorganisms with modified resistance to fungicides. The expression vector carrying the CcABCB5 gene can be used to transform microbial cells by conventional biological methods such as Ti plasmids, Ri plasmids, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc.

[0040] As a specific embodiment, the above-mentioned recombinant vector is the recombinant vector pPIC3.5-CcABCB5. The recombinant vector is a recombinant plasmid obtained by inserting a DNA fragment with a sequence of SEQ ID No. 2 between the restriction endonuclease cutting sites of BamH I and EcoR I of the starting vector pPIC3.5 and keeping the sequence unchanged.

[0041] The microorganism described herein can be yeast, bacteria, algae or fungi. Among them, the bacteria can be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. The microorganism can specifically be Coryneformis.

[0042] The present invention also provides the use of the above-mentioned protein CcABCB5 or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in any of the following:

[0043] U1) Use of the aforementioned protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in regulating the resistance of pathogens to fungicides;

[0044] U2) Use of the aforementioned protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for regulating the resistance of pathogens to fungicides;

[0045] U3) Use of the aforementioned protein or gene expression regulating substance or substance regulating the activity or content of the protein in cultivating pathogenic bacteria with improved resistance to fungicides;

[0046] U4) Use of the above-mentioned protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for pathogens with reduced resistance to fungicides.

[0047] In the above application, the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material can be any one of the following B1) to B7):

[0048] B1) a nucleic acid molecule encoding the protein described above;

[0049] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0050] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0051] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0052] C1) a nucleic acid molecule that inhibits, reduces or silences the expression of a gene encoding the protein described above;

[0053] C2) expressing the gene encoding the nucleic acid molecule described in C1);

[0054] C3) an expression cassette containing the coding gene described in C2);

[0055] C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3);

[0056] C5) A recombinant microorganism containing the coding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4).

[0057] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein CcABCB5.

[0058] The present invention also provides a method for regulating the resistance of pathogens to fungicides, comprising regulating the activity and / or content of the protein described above in the target pathogen, or / and the expression level of the gene encoding the protein, to regulate the resistance of the pathogen to fungicides.

[0059] In the above method, the regulation of the activity and / or content of the protein mentioned above in the target pathogen, or / and the expression level of the gene encoding the protein mentioned above includes introducing a recombinant vector in which the gene encoding the protein is knocked out into the recipient pathogen, thereby obtaining a pathogen with higher resistance to fungicides than the recipient pathogen; the encoding gene encodes the protein mentioned above.

[0060] The recombinant vector may be pEX-CcABCB5 or PCDNA3.1-CcABCB5d, etc.

[0061] In order to facilitate the identification and screening of transgenic cells, the recombinant expression vector used can be processed, such as adding genes encoding enzymes or luminescent compounds that can be expressed in microorganisms and can produce color reactions, antibiotic markers with resistance, or chemical agent resistance marker genes, etc. It is also possible to directly screen transgenic microorganisms by adversity without adding any selective marker genes.

[0062] The present invention also provides a method for cultivating pathogens with altered resistance to fungicides, comprising: inhibiting, reducing or silencing the expression level of the gene encoding the protein mentioned above in the target pathogen, or / and inhibiting, reducing or silencing the activity and / or content of the gene encoding the protein mentioned above, to obtain pathogens with improved resistance to fungicides.

[0063] The inhibiting of the activity and / or content of the gene encoding the protein may be knocking out the protein.

[0064] The inhibition, reduction or silencing of the expression of the nucleic acid molecule encoding the CcABCB5 protein in the target plant can be achieved by introducing a gene knockout vector targeting the nucleic acid molecule encoding the CcABCB5 protein into the target plant.

[0065] The gene knockout vector may be a ΔCcABCB5 knockout vector.

[0066] In the above application or method, the fungicide may be penthiopyrad, boscalid, kresoxim-methyl, myclobutanil, pyraclostrobin, difenoconazole, or fludioxonil. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the phylogenetic tree of 57 ABC proteins of Corynespora multilocus, Cc: Corynespora multilocus; Bc: Botrytis cinerea; Sc: Saccharomyces cerevisiae; FGSG: Fusarium spp.; Af: Aspergillus fumigatus.

[0068] Figure 2 This is the Motif, domain and gene structure analysis of the CcABC transporter protein, where A: motif; B: domain; C: gene structure analysis.

[0069] Figure 3 Chromosome positioning.

[0070] Figure 4 The expression levels of 57 CcABC genes under the treatment of trifloxystrobin, including (A): gene expression under trifloxystrobin treatment; (B): gene expression without trifloxystrobin treatment; (C): differential gene expression under the comparison analysis of trifloxystrobin; genes with differential expression under the three treatments are marked in red and blue. Strain 223 is a sensitive strain; strains 66, 71, 260 and 241 are resistant strains.

[0071] Figure 5 The construction and verification of the CcABC5 gene knockout mutant, where AC: Schematic diagram of the gene replacement strategy. The two gray fragments represent the left and right homologs of the CcABCB5 gene. The two black fragments represent the target genes. CcABCB5 and Hyg genes. A, CcABCB5 gene of parent strain 66; B, gene knockout vector constructed by fusion PCR; C, obtained based on homologous recombination. CcABCB5 gene knockout mutant; D, upstream fragment (973pb), Hyg gene (1755bp) and downstream gene (983bp); E, knockout vector; F, knockout mutant PCR verification, from top to bottom are upstream connection verification, downstream connection verification and homozygosity verification.

[0072] Figure 6 Biological characteristics of CcABCB5 knockout, including A, spore germination rate; B, pathogenicity; C, hyphal growth rate and colony morphology; D, stress; 66 is the parent strain, ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86 are knockout mutants constructed with 66 as the parent strain. *: indicates significant difference. DETAILED DESCRIPTION

[0073] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0074] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0075] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0076] The competent E. coli DH5α in the following examples was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0077] The pKHT plasmid in the following example contains a hygromycin B resistance gene, which has been recorded in: Zhu Fadi. Study on the resistance of Cucumber Corynespora cassiicola to boscalid and its mechanism [D]. Chinese Academy of Agricultural Sciences, 2019. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0078] The strains 66 and 71 in the following examples are resistant to trifloxystrobin, which has been recorded in: Sun B#, Zhou R#, Zhu G, Xie X, Chai A, Li L, Fan T, Zhang S, Li B*, Shi Y*. 2024. The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola. Pestic Biochem Physiol, 198: 105760. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0079] The G143A-HR strain in the following examples has been described in: Sun B#, Zhou R#, Zhu G, Xie X, Chai A, Li L, Fan T, Zhang S, Li B*, Shi Y*. 2024. The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola. Pestic Biochem Physiol, 198: 105760. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0080] Ampicillin sodium salt was purchased from Beijing Solebow Technology Co., Ltd. with a working concentration of 100 μg / mL.

[0081] Hygromycin B was purchased from Biomed and dissolved in sterile distilled water at a working concentration of 100 μg / mL.

[0082] Kanamycin was purchased from Beijing Solebao Technology Co., Ltd. with a working concentration of 100 μg / mL.

[0083] Pyraclostrobin: 98%, technical drug, Yulin Chengtaiheng Biotechnology Co., Ltd.

[0084] Trifloxystrobin: 97%, technical, Nantong Taihe Chemical Co., Ltd.

[0085] Kresoxim-methyl: 98%, technical, Jiangsu Heyutai Chemical Co., Ltd.

[0086] Azoxystrobin: 95%, technical drug, Yongnong Bioscience Co., Ltd.

[0087] Boscalid: 97%, technical, Shaanxi Meibang Pesticide Co., Ltd.

[0088] Penthiopyrad: 98.8%, technical product, Mitsui Chemicals AGRO Co., Ltd., Japan.

[0089] Difenoconazole: 95%, technical, Jiangsu Fengdeng Crop Protection Co., Ltd.

[0090] Fludioxonil: 96%, technical product, Liaoning Zhonghui Biotechnology Co., Ltd.

[0091] Spermidine: purchased from Sigma Aldrich.

[0092] Lyase: purchased from Sigma Aldrich.

[0093] The culture medium preparation method in the following examples is as follows:

[0094] PDA medium: glucose: 20 g, potato: 200 g, agar: 20 g, distilled water to 1 L, sterilize at 121°C and high temperature and high pressure.

[0095] YBA medium: yeast powder: 10 g, peptone: 10 g, sodium acetate: 20 g, agar: 15 g, distilled water to 1 L, sterilize at 121°C and high temperature and high pressure.

[0096] LB solid and liquid culture medium: tryptone: 10 g, yeast extract: 5 g, sodium chloride: 10 g, distilled water to 1 L, sterilized at 121°C and high temperature and high pressure, and agar is added to the solid culture medium.

[0097] RM medium: sucrose: 239.6 g, yeast powder: 0.5 g, enzymatic hydrolyzed casein: 0.5 g, agar: 16 g, distilled water to 1 L, sterilized at 121°C and high temperature and high pressure.

[0098] YEPD medium: glucose: 20 g, peptone: 10 g, yeast powder: 3 g, distilled water to 1 L, sterilized at 121°C.

[0099] SRM medium: sucrose: 342.3 g, yeast powder: 0.5 g, enzymatic hydrolyzed casein: 0.5 g, agarose: 12 g, distilled water to 1 L, sterilized at 121°C.

[0100] 0.7M NaCl buffer: NaCl 40.95g, distilled water to 1L, sterilize at 121℃.

[0101] STC solution: sorbitol: 145.74 g, Tris: 6.06 g, CaCl2: 5.55 g, distilled water to 1 L, sterilize at 121°C.

[0102] SPTC solution: PEG6000: 40g, STC to 1L, sterilize at 121℃.

[0103] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0104] Example 1: Identification of ABC proteins of Corynespora polytomata and construction of phylogenetic tree

[0105] 1. DNA and RNA extraction

[0106] DNA of Corynespora polymorpha strains 66, ΔCcABCB5-19, ΔCcABCB5-24, and ΔCcABCB5-86 was extracted using the CTAB method, and RNA was extracted using an RNA extraction kit. The detailed steps are as follows:

[0107] CTAB method for DNA extraction: Grind the sample, take 5-10g of mycelium into a 1.5mL centrifuge tube, put the centrifuge tube into a container filled with liquid nitrogen and let it stand for 30s, then use a grinding rod to grind the mycelium into powder. Lysis: Add 700μL CTAB, lyse at 55 degrees for 40min Extraction: Add 700μL chloroform, shake and mix, centrifuge at 12000rmp for 10min, take the supernatant; repeat this step once. Precipitation: Add 2 times the volume of anhydrous ethanol, centrifuge at 12000rmp for 10min, and discard the supernatant. Washing: Rinse with 75% alcohol, centrifuge at 12000rmp for 10min, and discard the supernatant. Repeat this step once. Dissolve DNA: Add 50-100μL ddH2O to dissolve, and store at -20 degrees for later use.

[0108] RNA extraction method refers to RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. DP432)

[0109] 2. ABC protein identification and phylogenetic tree construction

[0110] ABC transporter genes were identified based on the genome data of Corynespora polymyxinii (GCA_003016335.1). The ABC protein sequences of Corynespora polymyxinii were screened by local BLASTP with an E value set to 1.0E-10, using the ABC protein sequences of the B. cinerea genome (genbank: GCA_000143535.4 updated in February 2015), the Saccharomyces cerevisiae genome (genbank: GCA_000146045.2 updated in December 2014), and the Aspergillus fumigatus genome (genbank: GCA_000002655.1 updated in July 2005) as reference sequences. The candidate ABC proteins were then further screened using the CDD database (http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and the SMART (http: / / smart.embl-heidelberg.de / ) database, and sequences without ABC protein structure or protein sequence less than 100 aa were deleted from the database.

[0111] The phylogenetic tree was constructed using MEGA (version 7.0) software, and the algorithm and model were maximum likelihood (ML) and JTT model, respectively.

[0112] The results showed that in the genome of Corynespora polytomata, a total of 57 ABC transporter genes were identified by referring to the TMD and NBD domains ( Figure 1 ). Based on the ABC protein sequences of Saccharomyces cerevisiae, B. cinerea, Fusarium oxysporum, and Aspergillus fumigatus, a phylogenetic tree was constructed, which divided 57 CcABC transporters into 8 subfamilies and one YDR061W-like protein, including 2 ABCA, 19 ABCB, 9 ABCC, 2 ABCD, 2 ABCE, 5 ABCF, 16 ABCG, 1 ABCI, and 1 YDR061W-like protein. In all species, the most abundant subfamilies were ABCB and ABCG, while ABCI was the least abundant in each species. In Polymyxin, ABCB was the most abundant subfamily (Tables 1, 2, and 3).

[0113] Table 1. Candidate fungicide resistance-related proteins

[0114]

[0115] a: Protein function annotation based on the Uniprot database.

[0116] b: TMD: transmembrane protein; NBD: nucleic acid binding domain; N: N-terminus of ABC transporter.

[0117] Table 2 Functional annotation and sequence numbers of 57 CcABC transporters

[0118]

[0119]

[0120] 3. Motif, gene structure and physicochemical properties

[0121] The MEME Suite website (https: / / meme-suite.org / meme / tools / meme) was used to predict the motif of ABC protein, with the motif parameter set to 10 and other parameters as default values. The results were visualized using TBtool software (version 2.003, https: / / github.com / CJ-Chen / TBtools-II / releases). The subcellular localization of the protein was predicted using the WOLF PSORT (http: / / wolfpsort.hgc.jp / ) website. The TBtool software was used to analyze the physicochemical properties and gene structure of the protein and visualize it.

[0122] The results are as follows: Motif 1 and Motif 2 are ubiquitous in all subfamily members (CcABCA-CcABCI) ( Figure 2 ). However, the motifs of the same subfamily show high similarity, while the motifs between different subfamilies are significantly different. For example, Motifs 2, 6, 8, 10, 1, and 7 are common in the CcABCG subfamily, and Motifs 2, 5, 9, 1, and 4 are common in the CcABCB and CcABCC subfamilies.

[0123] The differences in CcABC gene structure indicate diversity between and within subfamilies. The number of exons varies from 1 to 18. For example, CcABCG16 proteins have the largest number of exons, while CcABCB14 subfamily has the fewest exons.

[0124] In addition, the conserved domains of CcABC transporters were predicted using CDD and Pafm online websites. The results showed that the domains of the same subfamily had high similarity, but the composition of the domains of different subfamilies was significantly different. Secondly, the domains of TMD and NBD domains in different subfamilies were significantly different. Finally, the number of domains in different subfamilies was different. For example, in the CcABCG subfamily, an additional domain (ABC_trans_N) was found, which was not present in other subfamilies.

[0125] Table 3. Physicochemical properties of 57 CcABC transporters

[0126]

[0127]

[0128] 4. Chromosome localization, gene annotation and protein interaction

[0129] Chromosome localization and visualization were performed based on the genome of Corynospora polymyxis using TBtools (version 2.003) software. Synteny analysis of Corynospora polymyxis was performed using the “advancedCircos” function in TBtools (version 2.003) software using gene annotation data. All CcABC transporters were annotated using the online website eggNOG-mapper (http: / / eggnog-mapper.embl.de / ). Protein-protein interaction (PPI) analysis was performed using the online STRING website service (http: / / string-db.org / ) with default parameters.

[0130] Detailed information on the annotation, chromosomal localization, subcellular localization, and physicochemical properties of CcABC transporters is shown in Tables 2 , 3 and Figure 3 Most CcABC transporters were annotated as ABC transporters, P-loops containing nucleases, heavy metal resistance proteins, and drug resistance proteins. A few CcABC transporters were annotated as translation factors, such as the CcABCE1 gene, which was annotated as a translation initiation factor. The diversity of CcABC transporter annotations suggests the diversity of their functions.

[0131] This paper screened out 12 candidate resistance-related proteins, including 7 CcABCBs proteins (CcABC1, CcABC10, CcABC4, CcABC5, CcABC6, CcABC8 and CcABC9), 1 CcABCC protein (CcABCC5) and 4 CcABCG proteins (CcABCG5, CcABCG6, CcABCG8 and CcABCG15). Among them, CcABCB5 protein was differentially expressed in the presence or absence of oxime induction ( Figure 4 (A)-(B)), and the expression level of CcABCB5 gene in resistant strains was significantly higher than that in sensitive strains ( Figure 4 (C)).

[0132] Since the genome of Corynespora polymyxinii is not spliced ​​at the chromosome level, the CcABC transporters were localized at the scaffold level. The results showed that 57 CcABC transporters were localized in 12 scaffolds, but the number of transporters on each scaffold was different, which was 12 on scaffold_1, 12 on scaffold_2, 4 on scaffold_4, 3 on scaffold_6, 2 on scaffold_16, 1 on scaffold_8, 1 on scaffold_12, 1 on scaffold_13, and 1 on scaffold_15.

[0133] The number of amino acid residues ranged from 286 to 1745; the molecular weight ranged from 31.83 to 193.09 kDa; the pI ranged from 5.30 to 9.46; the GRAVY ranged from -0.624 to 0.291; and the instability index ranged from 30.41 to 50.69. The detailed information on the physicochemical properties of the CcABC transporter is shown in Table 3.

[0134] Example 2: Construction of CcABC5 gene deletion mutant

[0135] The CcABCB5 gene in the genomic DNA of the CcABC5 protein is shown in SEQ ID No. 1 of the sequence listing, the coding sequence of the CcABC5 gene is shown in SEQ ID No. 2 of the sequence listing, and the encoded amino acid sequence is the protein CcABC5 shown in SEQ ID No. 3 of the sequence listing.

[0136] 1. Construction of CcABC5 gene deletion vector

[0137] Double-joint PCR technology was used to construct gene deletion mutants. The vector construction strategy was as follows: Figure 5 As shown in AC.

[0138] 1) The upstream 973bp (fragment A), downstream 983bp (fragment B) and selection marker gene Hyg 1755bp (fragment C) of CcABC5 gene were amplified and purified respectively, and then the three fragments were fused at a molar ratio of 1:3:1 by fusion PCR. The connection order from left to right was: fragment A-fragment B-fragment C, and the connection method of the three fragments was: fusion PCR. Finally, the fusion product template was used for large-scale nested PCR amplification to obtain the gene deletion vector ΔCcABCB5, and a 3711bp vector was obtained (the vector nucleotide sequence is SEQ ID No. 4 in the sequence list). The primer information used for construction is shown in Table 4.

[0139] The fusion vector was sequenced using the 6071-up-F / 6071-down-R primer set, and sequencing confirmed that there were no other nucleic acid mutations and that it could be used for subsequent experiments.

[0140] 2) The constructed deletion vector ΔCcABCB5 was transformed into the polymyxin Bacillus strain 66 according to the protoplast transformation method to obtain the recombinant polymyxin Bacillus ΔCcABC5. The specific protoplast transformation operation steps are shown below.

[0141] Protoplast genetic transformation method:

[0142] Collection of mycelium: After activating the multi-homocybin fungus plate, transfer it into YEPD liquid culture medium and shake culture at 28°C for 24h-48h.

[0143] Lysis: Prepare 15 mg / mL lysing enzyme, filter through three layers of sterile lens paper to obtain mycelial pellets (1-3 mm), and rinse three times with 0.7 M NaCl buffer. Transfer the rinsed mycelial pellets into the enzyme solution in a triangular bottle, and then place in a shaker at 80 rpm and 30°C for lysis for 2-4 hours.

[0144] Protoplast collection: use three layers of lens paper to filter the lysis solution to obtain a protoplast suspension, centrifuge at 1200 rpm for 5 min, discard the supernatant, rinse again with 0.7 M NaCl buffer, centrifuge at 1200 rpm for 5 min, and discard the supernatant; use STC solution to resuspend the protoplasts, centrifuge at 1200 rpm for 5 min, discard the supernatant, and the precipitate is the protoplasts; use STC solution to resuspend, shake carefully after adding to prevent the formation of lumps, adjust its concentration to 2-3×108 cells / mL, and add SPTC solution at a ratio of STC:SPTC=4:1.

[0145] PEG-mediated transformation: Take 200 μL of protoplast transformation tube, add 4 ng of vector fragment and 10 μL of spermidine, mix gently, and place on ice for 1 hour (mix intermittently every 20 minutes); then add 1 mL of SPTC solution, shake gently, and place at room temperature for 1 hour; then add RM regeneration medium cooled to 55°C to the transformation tube, shake well and pour into a plate, and culture at 28°C for 12 hours to grow new cell walls. Then cover the plate with SRM medium, in which antibiotics are added, and culture at 28°C for 4-10 days.

[0146] Antibiotic verification: Pick a single colony from the SRM plate and place it on a PDA plate containing antibiotics. After culturing at 28°C for 4-5 days, transfer it to a PDA plate containing hygromycin for 6-10 generations. Transformants that can grow normally may be the target transformants.

[0147] 3) PCR verification: In order to verify the ΔCcABC5 knockout mutant, the upstream connection, downstream connection and homozygosity of the CcABC5 gene were verified respectively. The specific verification method is: use primers Det-6071-up-F (5'-CCCAAGGCCCAAATCTAGTC-3') and Det-6071-up-R (5'-AGTACTCGCCGATAGTGGAA-3') to verify the upstream connection; use primers Det-6071-down-F (5'-AAGTTATCGTGCACCAAGCA-3') and Det-6071-down-R (5'-AGCATGCAATGACGCTGATA-3') to verify the downstream connection; use primers Det-6071-hom-F (5'-TTTGGTGGCCAGAACTTCGT-3') and Det-6071-hom-R (5'-TTCGCCAAGCAGATTGAGGT-3') to verify the homozygosity.

[0148] The results showed that: Figure 5 D, E and F) in total, three ΔCcABC5 knockout mutants were obtained, namely ΔCcABC5-19, ΔCcABC5-24 and ΔCcABC5-86 ( Figure 5(ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86). In the upstream connection detection of CcABC5 gene, a fragment of 1788bp was amplified in the knockout mutant recombinant Coryneformis ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86, but no amplification was found in the parent strain 66, indicating that the knockout vector was correctly connected to the corresponding position; in the downstream connection detection, a fragment of 1692bp was amplified in the knockout mutant recombinant Coryneformis ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86, but no amplification was found in the parent strain 66, indicating that the knockout vector ΔCcABCB5 was correctly connected to the corresponding position; in the homozygosity detection of gene knockout, only a single fragment of 3091bp was amplified in the knockout mutant recombinant Coryneformis ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86, and no other bands were amplified, indicating that the CcABC5 gene had been knocked out.

[0149] Table 4. Primer information used in the present invention

[0150]

[0151]

[0152] Example 3. Phenotypic analysis of CcABC5 gene deletion mutants

[0153] The strains to be tested included CcABC5 gene deletion mutants ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86, and the parent strain 66 was used as a control.

[0154] 1. Study on spore production

[0155] Use a 1cm borer to punch 5 bacterial cakes at the edge of the colony of the multi-homed Coryneformis, place them in a 10mL centrifuge tube, add 3mL of 0.1% Tween water and 10 glass beads with a diameter of 5mm, and oscillate on a vortex shaker for 2min to obtain a suspension of multi-homed Coryneformis spores. Use a pipette to draw 20μL, drop it on the scale line of the hemocytometer, and cover it with a coverslip for counting.

[0156] Spore germination ability of multi-primary coryneformis: After measuring the spore production on the plate, use 0.1% Tween water to elute the multi-primary coryneformis spores from the plate, filter the plate with gauze to prepare a multi-primary coryneformis spore suspension, and draw 80 μL of the spore suspension and drop it into the groove of the double concave glass slide. Set 6 replicates for each strain, and culture at 28°C for 4-6 hours. Investigate 100 multi-primary coryneformis spores under an optical microscope, and use the germ tube length as 1 / 2 of the spore length as the germination standard, and record the number of multi-primary coryneformis spores that germinate.

[0157] 2. Pathogenicity studies

[0158] Cucumber leaves of the same leaf age were collected, and wounds were made by piercing the leaves with sterilized toothpicks. The wounds were inoculated with bacterial cakes with a diameter of 5 mm, and cultured in a light incubator with a relative humidity and temperature of 90% and 28°C alternating with darkness for 5 days. The diameter of the lesions was then determined by the cross method.

[0159] 3. Osmotic stress and cell wall stress assay

[0160] 1 M glucose, 1 M NaCl and 1.2 M KCl were used as osmotic stress reagents, and 0.3 mg mL -1 Congo red was used as a cell wall stress reagent. A 5 mm punch was used to punch out the bacterial cake at the edge of the colony, and a sterilized toothpick was used to inoculate the bacterial cake onto a PDA plate containing different reagents. The plate without reagent was used as a blank control, and cultured at 28°C for 7 days. The colony diameter was determined by the cross method and the inhibition rate was calculated.

[0161] The results showed that the CcABCB5 gene is involved in regulating the colony color, spore production and germination, pathogenicity, and osmotic and cell wall stress of Coryneformis multilocus. Figure 6 (in AD).

[0162] 1) Mycelial growth and colony morphology: There was no significant difference in mycelial growth rate between the CcABCB5 gene deletion mutants ΔCcABCB5-19, ΔCcABCB5-24, and ΔCcABCB5-86 and the parent strain 66. However, there was a significant difference in colony color: the front and back colonies of the parent strain 66 were both dark green, while the front colonies of the CcABCB5 deletion mutants ΔCcABCB5-19, ΔCcABCB5-24, and ΔCcABCB5-86 were white and the back colonies were light red.

[0163] 2) Spore production and germination: The spore production of CcABCB5 deletion mutants was significantly reduced, ranging from 3.07% to 36.65% of the parent strain. However, CcABCB5 gene deletion mutants ΔCcABCB5-19, ΔCcABCB5-24, and ΔCcABCB5-86 showed higher spore germination rates, ranging from 218.92% to 227.94% of the parent strain.

[0164] 3) Pathogenicity: The pathogenicity of the CcABCB5 deletion mutants ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86 was significantly stronger than that of the parental strain 66, which was 1.70-2.06 times that of the parental strain.

[0165] 4) Osmotic and cell wall stress: Under osmotic stress of 1M KCl and 1M NaCl, the mycelial inhibition rates of CcABCB5 deletion mutants ΔCcABCB5-19, ΔCcABCB5-24, and ΔCcABCB5-86 were 1.21-1.47 times higher than that of the parent strain 66. However, no significant difference was observed under osmotic stress of 1M glucose. Under cell wall stress caused by Congo red, CcABCB5 deletion mutants ΔCcABCB5-19, ΔCcABCB5-24, and ΔCcABCB5-86 showed higher sensitivity than the parent strain 66.

[0166] 4. Fungicide sensitivity determination

[0167] The mycelial growth rate method was used to determine the sensitivity of the deletion mutants ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86 to fungicides.

[0168] Activation of mutant strains: ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86 bacterial blocks were picked from the strains stored in a 4°C refrigerator for activation. After growing for 3 days, mycelial blocks were picked from the edge of the ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86 colonies for propagation culture for 5 days to obtain ΔCcABCB5-19, ΔCcABCB5-24 and ΔCcABCB5-86 bacterial agents for use.

[0169] Preparation of fungicides: dissolve pyraclostrobin, kresoxim-methyl, myclobutanil, boscalid, penthiopyrad, difenoconazole and fludioxonil in DMSO, prepare the mother solution and store it at 4℃ for later use. (accurate to one ten-thousandth)

[0170] Preparation of fungicide-containing plates: Take the required fungicide mother solution and add it into a PDA flask that has been melted and cooled to about 55°C, shake well, pour into a culture dish with a diameter of 6 mm, dry it and set it aside, and repeat it 3 times.

[0171] Inoculation: Use a 5mm borer to punch a bacterial cake at the edge of the colony of the above-cultured cucumber multi-homocybin fungus (C. cassiicola), pick up the bacterial cake with a sterilized toothpick, and stick it on the center of the drug-containing plate with the mycelium side facing down.

[0172] Investigation: Culture at 28℃ for 4-5 days, use the cross method to investigate and record the diameter of the colony.

[0173] Inhibition rate = 1-((net colony diameter at drug concentration / net colony diameter of blank control) x 100%).

[0174] EC 50Calculation: The EC value was calculated using the Probit function in SPSS (version 26) software. 50 value.

[0175] The concentration gradients of the fungicides were as follows: pyraclostrobin (0.1, 1, 2.5, 10, 20 μg / mL), kresoxim-methyl (5, 50, 100, 200, 400 μg / mL), azoxystrobin (0.2, 1, 5, 25, 100 μg / mL), boscalid (0.1, 1, 5, 10, 30 μg / mL), isopyrazam (0.1, 1, 5, 10, 30 μg / mL), difenoconazole (0.1, 2, 6, 10, 20 μg / mL), and fludioxonil (0.1, 0.5, 1, 5, 10 μg / mL).

[0176] The results showed that the CcABCB5 transporter was involved in regulating the sensitivity of C. cassiicola to fungicides (Table 5), but there were significant differences in the sensitivity to different fungicides after knocking out the CcABCB5 gene. According to the EC 50 and resistance factor, the changes in sensitivity to different fungicides were divided into three groups: one group, enhanced sensitivity, isopyrazam: 0.17 < RFs < 0.66; boscalid: 0.34 < RFs < 0.95; kresoxim-methyl: 0.11 < RFs < 0.50; the second group, decreased sensitivity, azoxystrobin: 1.15 < RFs < 4.60; pyraclostrobin: 5.08 < RFs < 9.45; difenoconazole: 2.80 < RFs < 8.70; the third group, no change, fludioxonil: 1.02 < RFs < 1.73. Where RF represents the resistance factor, and the calculation formula is RF = EC 50 (mutant) / EC 50 (parent strain).

[0177] Table 5 Sensitivity of ΔCcABCB5 knockout mutants to different fungicides

[0178]

[0179]

[0180] Note: In the table, RF represents the resistance factor, and EC 50 represents the median inhibitory concentration, and the values are the mean ± standard error; the same capital letters in the same column represent no significant difference in RF among the tested strains; the same lowercase letters in the same column represent no significant difference in EC 50 among the tested strains.

[0181] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application, characterized in that: The application is any of the following: U1) Use of a substance that regulates the expression of a protein encoding gene or a substance that regulates the activity or content of the protein in regulating the resistance of plant pathogens to fungicides; U2) Use of a substance that regulates the expression of a protein encoding gene or a substance that regulates the activity or content of the protein in the preparation of a product that regulates the resistance of plant pathogens to fungicides; The substance regulating the expression of the protein encoding gene or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material is a gene deletion vector Δ CcABCB5 , the gene deletion vector Δ CcABCB5 The nucleotide sequence is SEQ ID No. 4 in the sequence list; The plant pathogen is Corynespora polytomata.

2. The use according to claim 1, characterized in that: The fungicide is pyraclostrobin, kresoxim-methyl, myclobutanil, boscalid, penthiopyrad or / and difenoconazole.

3. Use of a substance that regulates the expression of a protein encoding gene or a substance that regulates the activity or content of the protein in cultivating plant pathogens with improved resistance to fungicides; The substance regulating the expression of the protein encoding gene or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material is a gene deletion vector Δ CcABCB5 , the gene deletion vector Δ CcABCB5 The nucleotide sequence is SEQ ID No. 4 in the sequence list; The plant pathogen is Corynespora polytomata.

4. The use according to claim 3, characterized in that: The fungicide is pyraclostrobin, myclobutanil or / and difenoconazole.

5. A method for regulating the resistance of pathogens to fungicides, characterized in that: Including regulating the content of protein in the target pathogen or the expression of the gene encoding the protein to regulate the resistance of the pathogen to the fungicide; The regulation of the protein content in the target pathogen or the expression of the protein encoding gene is to inhibit, reduce or silence the expression of the protein encoding gene in the recipient pathogen, thereby obtaining a pathogen with higher resistance to the fungicide than the recipient pathogen; The protein is a protein whose amino acid sequence is shown in SEQ ID No.3; The pathogen is Corynespora multilocus.

6. The method according to claim 5, characterized in that: The fungicide is pyraclostrobin, myclobutanil or / and difenoconazole.

7. The method according to claim 5, characterized in that: The recipient pathogen is Corynespora polymorpha strain 66; The fungicide is pyraclostrobin, myclobutanil or / and difenoconazole.

8. A method for cultivating pathogenic bacteria with modified resistance to fungicides, comprising: Inhibiting, reducing or silencing the expression of a gene encoding a protein in a target pathogen or reducing the content of the protein to obtain a pathogen with improved resistance to fungicides; The protein is a protein whose amino acid sequence is shown in SEQ ID No.3; The pathogen is Corynespora multilocus; The fungicide is pyraclostrobin, myclobutanil or / and difenoconazole.

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