Application of xoFabF1 protein and its encoding gene in regulating virulence of xanthomonas oryzae
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF PLANT PROTECTION JIANGXI ACAD OF AGRI SCI
- Filing Date
- 2026-04-01
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168563A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the XoFabF1 protein and its encoding gene in regulating the virulence of rice bacterial blight pathogen. Background Technology
[0002] Rice is a core food crop for ensuring global food supply, and its stable and high yields are directly related to global food security and people's livelihoods. Rice bacterial blight is caused by the fungus *Bacillus oryzae* (rice bacterial blight pathogen). Xanthomonas rice pv. rice abbreviation Xoo Rice bacterial blight is a devastating bacterial disease caused by the pathogen. It spreads rapidly and infects a wide area, causing significant yield reductions and, in severe cases, complete crop failure, resulting in incalculable economic losses to the rice industry. Currently, conventional control methods for rice bacterial blight mainly rely on chemical pesticide spraying and the breeding of disease-resistant varieties. However, long-term and excessive use of chemical pesticides not only leads to ecological problems such as pesticide residues and soil and water pollution, but also promotes the rapid development of drug resistance through genetic variation in the pathogen, causing a continuous decline in the effectiveness of existing control agents. Simultaneously, the resistance of resistant varieties is easily lost due to changes in the pathogen population, further exacerbating the difficulty of disease control. Therefore, in-depth analysis of the physiological and biochemical characteristics and molecular pathogenic mechanisms of rice bacterial blight pathogens, and the identification of key pathogenic regulatory targets, are the core prerequisites for developing efficient, green, and eco-friendly disease control technologies, and the key to overcoming the current control dilemma.
[0003] Fatty acid synthesis is a core metabolic pathway for bacteria to maintain growth and reproduction and complete life activities. 3-Ketoacyl-ACP synthase II, as a key rate-limiting enzyme in the elongation stage of bacterial fatty acid synthesis, participates in regulating the elongation and synthesis of bacterial fatty acid chains, playing an important role in bacterial growth and development, metabolic homeostasis, and pathogenicity regulation. This type of enzyme is widely distributed and highly conserved in pathogens of the genus Xanthomonas. Previous studies have found that the causal agent of rice bacterial blight exists in *Xanthomonas auricula-judae*, encoding the XoFabF1 protein. XofabF1 The gene encodes a protein composed of 411 amino acids, which has a homology of 58.7% with Escherichia coli 3-ketoacyl-ACP synthase II and possesses typical structural features of 3-ketoacyl-ACP synthase II. It is speculated that the gene plays an important role in the life activities of rice bacterial blight pathogen.
[0004] Currently, research on 3-ketoacyl-ACP synthase II largely focuses on fatty acid metabolism regulation in model bacteria. However, systematic research reports on the core aspects of XoFabF1 protein in rice bacterial blight pathogens, including its biological function, regulatory role in pathogenicity and related physiological traits, and molecular regulatory mechanisms, are lacking. This leaves a significant gap in functional analysis and application development. Existing research cannot clearly define… XofabF1 The specific regulatory effects of genes in the pathogenesis of rice bacterial blight pathogens are not well understood, and it is difficult to discover new disease control targets based on these genes, which greatly limits the development of green control technologies targeting key pathogenic genes of the pathogen. Therefore, research is being conducted... XofabF1 Functional studies of the gene, clarifying its regulatory mechanism on the virulence, growth, reproduction and pathogenic factors of rice bacterial blight, have important theoretical research value and practical application potential, and can provide a new theoretical basis and technical direction for the green control of rice bacterial blight. Summary of the Invention
[0005] Based on this, the present invention aims to provide an application of XoFabF1 (3-ketoacyl-ACP synthase II) protein and its encoding gene in regulating the virulence of rice bacterial blight pathogen.
[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides, on the one hand, the XoFabF1 protein and its encoding gene in regulating the role of rice bacterial blight pathogen (… Xanthomonas oryzae pv. rice Applications in virulence: The amino acid sequence of the XoFabF1 protein is shown in SEQ ID NO.1.
[0007] Preferably, in the above applications, the nucleotide sequence of the gene encoding the XoFabF1 protein is shown in SEQ ID NO.2.
[0008] Preferably, in the above applications, the reduction of XoFabF1 protein activity or the knockout or knockdown of the gene encoding XoFabF1 protein is used to reduce the virulence, growth rate, motility and extracellular polysaccharide synthesis of rice bacterial blight pathogen.
[0009] Another aspect of the present invention provides a rice bacterial blight pathogen Δ XofabF1 Mutant strains or their suspensions or freeze-dried powders, rice bacterial blight pathogen Δ XofabF1 Mutant strains were created by knocking down or eliminating the pathogen of rice bacterial blight. XofabF1 Gene acquisition.
[0010] In another aspect, the present invention provides the above-mentioned rice bacterial blight pathogen Δ XofabF1 The method for constructing mutant strains is characterized by comprising: using a suicide vector as a vector, primer amplification to obtain... XofabF1Upstream and downstream gene fragments are digested with two enzymes to... XofabF1 The upstream and downstream gene fragments were inserted into an enzyme digestion vector to construct the pK18-XofabF1 homologous recombination vector, which was then transformed into a wild-type strain of *Bacillus oryzae* var. *rice* and cultured to obtain *Bacillus oryzae* Δ. XofabF1 Mutant strain.
[0011] Preferably, in the above construction method, the suicide vector is pK18mobsacB.
[0012] In another aspect, the present invention provides XofabF1 The application of genes as targets in screening drugs to control rice bacterial blight pathogens: the drugs can reduce the expression level of the XoFabF1 protein encoding gene of rice bacterial blight pathogens or inhibit the activity of XoFabF1 protein.
[0013] The beneficial effects of this invention include at least the following: This invention constructs a fungus that causes bacterial blight in rice. XofabF1 Gene-deleted strains XofabF1 Gene function was studied. The study found that knockout… XofabF1 Gene (△) XofabF1 Significantly reduced Xoo The pathogenicity, growth rate, extracellular polysaccharide (EPS) synthesis, and motility of *Bacillus oryzae* were observed. This indicates that 3-ketoacyl-ACP synthase II XoFabF1 positively regulates the virulence, growth rate, motility, and extracellular polysaccharide synthesis of *Bacillus oryzae*, making this invention of significant application value. Attached Figure Description
[0014] Figure 1 For △ XofabF1 PCR identification of mutant strains; lane M represents DNA marker DL2000 (2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); lane 1 represents the amplified fragment of the wild-type strain of *Bacillus thuringiensis*, the causal agent of rice bacterial blight; lane 2 represents the Δ... XofabF1 Amplified fragments of mutant strains; Figure 2 Wild-type strain and △ XofabF1 The infection experiment of mutant strains on the leaves of "Nipponbare" rice; Figure 3 Wild-type strain and △ XofabF1 Analysis of statistical results on lesion length of mutant strains; Figure 4 Wild-type strain and △ XofabF1 Growth curve of the mutant strain; Figure 5 Wild-type strain and △ XofabF1 The extracellular polysaccharide production of the mutant strain; Figure 6 Wild-type strain and △ XofabF1 Motility assay of mutant strains. Detailed Implementation
[0015] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.
[0016] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.
[0017] In a first aspect, embodiments of the present invention provide the XoFabF1 protein and its encoding gene in regulating the role of rice bacterial blight pathogen (… Xanthomonas oryzae pv. rice Applications in virulence: The amino acid sequence of the XoFabF1 protein is shown in SEQ ID NO.1.
[0018] It should be noted that rice bacterial blight is a devastating bacterial disease caused by *Bacillus oryzae*, which severely restricts high and stable rice yields. Identifying key targets for pathogen virulence regulation is crucial for achieving green disease control. The XoFabF1 protein is a key functional protein in the fatty acid synthesis pathway of *Bacillus oryzae*, participating in cell membrane lipid synthesis, maintenance of intracellular metabolic homeostasis, and secretion of pathogenic factors. This invention is the first to demonstrate that this protein is a key factor regulating the virulence of *Bacillus oryzae*, and its expression level directly determines the pathogen's infectivity, colonization efficiency, and the severity of pathogenic symptoms. This provides a novel core target for elucidating the virulence regulation mechanism of *Bacillus oryzae* and developing targeted control drugs, filling a research gap in the regulation of fatty acid synthesis-related virulence factors of this pathogen.
[0019] Additionally, the sequence shown in SEQ ID NO.1 is as follows: MSRRVVVTGMGIVSPLGNDLATSWDGIVHGRSGIGPITQIDASQFTTKIAGEIKNFDPTLFVSAKDVKKMDSFIHYGLGASFMALDDSGLEIDDSNAERVGAI LGSGIGGLLGIEEQTIKFHEGGARKISPFYVPSTIINMLPGQVSLIKGLKGPTFSAVSACATSNHSIGTAMRMIQHGDADVMLAGGAERGSSPGSVGGFCAMK AMSTRNDDPTGASRPWDKQRDGFVLGDGAGVLVLEEYEHAKARGARIYAELVGFGASSDAFHMTAPSEDGEGAARSMAAAMRDAKLNPEQIGYLNAHGTSTPLGDLAETIAMKRALGDHAYKTMVSSTKSMTGHLLGAAGGVEAIFSVMALHTGIIPPTINLEEPSEGCDLDYVPNVAREVQVDAVMSNGFGFGGTNGTLVFKRV.
[0020] In some specific examples, in the applications described above, the nucleotide sequence of the gene encoding the XoFabF1 protein is shown in SEQ ID NO. 2.
[0021] It should be noted that, XofabF1 The gene is a structural gene encoding the XoFabF1 functional protein. Its nucleotide sequence, verified by whole-genome sequencing and cloning sequencing, is a complete match with the gene sequence of wild-type *Bacillus oryzae* strains of rice, with no base mutations, deletions, or insertions. It can be precisely transcribed and translated into the XoFabF1 protein, which has complete virulence regulatory functions. This gene sequence is highly conserved, showing no significant polymorphism in different pathogenic strains of *Bacillus oryzae*. Knockout, knockdown, and targeted inhibition of this gene sequence can stably regulate XoFabF1 protein expression, thereby achieving precise regulation of pathogen virulence and providing accurate sequence evidence for subsequent gene editing and target drug screening.
[0022] Additionally, the sequence shown in SEQ ID NO.2 is as follows:
[0023] In some specific examples, the above applications include reducing XoFabF1 protein activity or knocking out or down the gene encoding XoFabF1 protein in reducing the virulence, growth rate, extracellular polysaccharide synthesis, or motility of rice bacterial blight pathogen.
[0024] It should be noted that the functional verification experiments show that, XofabF1 Gene expression levels and XoFabF1 protein activity were positively correlated with the virulence, growth rate, extracellular polysaccharide synthesis, and motility of rice bacterial blight pathogen. These factors were mitigated through gene knockout, RNA interference, and antisense oligonucleotide techniques to knock down / remove the pathogen. XofabF1 Genetic inhibition, or the use of small molecule inhibitors and protein inactivators to reduce the activity of XoFabF1 protein, can significantly block the fatty acid synthesis pathway of the pathogen, disrupt the integrity of the pathogen's cell membrane, inhibit the colonization, proliferation, and spread of the pathogen in rice plants, and greatly reduce the pathogen's pathogenicity, resulting in shorter rice lesion lengths and a significant decrease in the disease index. This application overcomes the shortcomings of traditional chemical pesticides, such as poor targeting and easy development of resistance, and provides a novel molecular intervention strategy for the green control of rice bacterial blight.
[0025] Secondly, embodiments of the present invention provide a rice bacterial blight pathogen Δ XofabF1 Mutant strains or their suspensions or freeze-dried powders, rice bacterial blight pathogen Δ XofabF1 Mutant strains were created by knocking down or eliminating the pathogen of rice bacterial blight. XofabF1 Gene acquisition.
[0026] It should be noted that Δ XofabF1 Mutant strains are produced by knocking out or reducing the levels of the wild-type strain of *Bacillus thuringiensis*, the causal agent of rice bacterial blight, through gene editing technologies such as homologous recombination and CRISPR / Cas9. XofabF1 The gene-constructed defective strain was identified by PCR, verified by sequencing, and its protein expression was detected, confirming that the strain contained... XofabF1 The gene cannot be transcribed normally, and there is no functional XoFabF1 protein expression. This mutant strain retains the basic growth activity of the wild-type strain, but its virulence is significantly reduced. It cannot infect healthy rice plants or can only cause very mild symptoms, thus possessing the core characteristics of a biocontrol strain. Its suspension and freeze-dried powder formulations can stably preserve the activity of the strain, making it convenient for field application, transportation and storage, and suitable for the actual needs of rice field disease control.
[0027] Thirdly, embodiments of the present invention provide a method for treating the above-mentioned rice bacterial blight pathogen Δ XofabF1 The method for constructing mutant strains includes: using a suicide vector as a vector, primer amplification, and obtaining the mutant strain. XofabF1 Upstream and downstream gene fragments are digested with two enzymes to... XofabF1The upstream and downstream gene fragments were inserted into an enzyme digestion vector to construct the pK18-XofabF1 homologous recombination vector, which was then transformed into a wild-type strain of *Bacillus oryzae* var. *rice* and cultured to obtain *Bacillus oryzae* Δ. XofabF1 Mutant strain.
[0028] It should be noted that this construction method relies on homologous recombination technology to target and knock out... XofabF1 The gene construct has a mature and stable process, a clear genetic background, and no risk of random insertion of exogenous resistance genes. It is first amplified using specific primers. XofabF1 Homologous arm fragments upstream and downstream of the gene were directionally ligated into a suicide vector after double enzyme digestion to obtain a homologous recombination vector. This vector was then introduced into wild-type rice bacterial blight pathogen via electroporation and conjugation transfer. The vector's suicide characteristics and homologous recombination mechanism were utilized to replace the wild-type pathogen in the genome. XofabF1 After resistance selection, PCR identification, and sequencing verification, the gene was found to be genetically stable Δ. XofabF1 The mutant strain has a high success rate and strong reproducibility, making it suitable for large-scale construction in the laboratory.
[0029] In some specific examples, the suicide vector in the above construction method is pK18mobsacB.
[0030] It should be noted that pK18mobsacB is a commonly used suicide vector for gene knockout in Gram-negative bacteria, possessing... sacB Reverse selection markers and kanamycin resistance markers are adapted for genetic manipulation of *Bacillus oryzae*, the causal agent of rice bacterial blight. This vector cannot replicate autonomously within *Bacillus oryzae* and can only integrate into the genome through homologous recombination. Subsequent reverse selection with sucrose and kanamycin resistance can efficiently eliminate wild-type strains that have not undergone homologous recombination, precisely selecting for... XofabF1 Gene knockout positive mutants significantly improve the screening efficiency of mutants and ensure Δ XofabF1 The genetic purity and stability of the mutant strain are the optimal vector selection for constructing the mutant strain.
[0031] Fourthly, embodiments of the present invention provide a XofabF1 The application of genes as targets in screening drugs to control rice bacterial blight pathogens: the drugs can reduce the expression level of the XoFabF1 protein encoding gene of rice bacterial blight pathogens or inhibit the activity of XoFabF1 protein.
[0032] It should be noted that, XofabF1 As an essential gene for regulating the virulence of rice bacterial blight pathogen, this gene is an ideal target for screening targeted antibacterial drugs. Using this gene as a target, a high-throughput drug screening system can be established, and targeted screening can inhibit... XofabF1Small molecule compounds that induce gene transcription, reduce XoFabF1 protein expression, or block protein activity, antimicrobial peptides, and nucleic acid drugs, etc. The screened drugs can specifically target pathogens. XofabF1 This gene does not affect the normal growth and metabolism of rice. It has strong targeting and significant antibacterial effect, and is not likely to induce drug resistance in pathogens. It breaks through the drawbacks of the broad-spectrum bactericidal effect of traditional chemical pesticides, and provides a core direction for the development of efficient, specific and green new drugs for the prevention and control of rice bacterial blight. It has extremely high scientific research and industrial application value.
[0033] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.
[0034] Example 1: Construction of ΔXofabF1 gene knockout strain The primer sequences used in the following examples are shown in Table 1.
[0035] Table 1 Primer sequences used in Example 1
[0036] (1) Amplification of the target gene fragment This study used the suicide vector pK18mobsacB to construct the rice bacterial blight pathogen (… Xanthomonas rice pv. rice , Xoo )Δ XofabF1 Mutant strain. Using the genome of the wild-type strain of *Bacillus oryzae*, the causal agent of rice bacterial blight, as a template, and following the methods reported in the literature (Yan et al. 2023. 3-Ketoacyl-ACP synthase III FabH1 isessential for branched-chain DSF family signals in...), mutant strains were developed. Xanthomonas rice pv. rice Phytopathology Research, 5, 26.) Amplification using the pK18-XofabF1 P1 / pK18-XofabF1 P2 primer pair. XofabF1 Upstream fragment 1 of the gene was amplified using the pK18-XofabF1 P3 / pK18-XofabF1P4 primer pair. XofabF1 Downstream segment 2 of the gene. Then... XofabF1 Using upstream fragment 1 and downstream fragment 2 of the gene as templates, and pK18-XofabF1 P1 / pK18-XofabF1 P4 as primer pairs, PCR amplification was performed to obtain... XofabF1 3. Upstream and downstream combination fragments of the gene.
[0037] (2) Construction of pK18-ΔXofabF1 homologous recombination vector pass Christmas I and Hind Restriction endonucleases were used to double-digest the amplified upstream and downstream combined fragment 3 and the pK18mobsacB vector, respectively. The digested upstream and downstream fragment 3 and the pK18mobsacB vector backbone were then recovered. The upstream and downstream fragment 3 were ligated to the pK18mobsacB vector using T4 DNA ligase (vector:fragment 3 = 1:3), thus... XofabF1 The upstream and downstream combination fragment 3 of the gene was directionally ligated into the pK18mobsacB plasmid. After the ligation product was transformed into competent cells, it was plated on LB resistant plates containing 30 μg / mL kanamycin (Kan) and cultured. Positive transformants were screened and sequenced to verify the results, and the pK18-ΔXofabF1 homologous recombination vector was constructed.
[0038] (3) Obtaining a recombinant strain The pK18-ΔXofabF1 recombinant plasmid, which was verified by sequencing, was transformed into *Bacillus oryzae*, the pathogen of rice bacterial blight, via electroporation. Xoo In competent cells, recombinant strains were screened once using sugar-free NA (3g beef extract, 5g peptone, 1g yeast extract, 15g agar powder per liter) resistance plates containing 30 μg / mL kanamycin (Kan). The selected recombinants were verified by PCR using primers pK18-XofabF1 P1 and pK18-XofabF1 P4. Strains showing two bands in the PCR amplification product with the expected band size and correct sequencing verification were identified as *Bacillus thuringiensis*, the pathogen of rice bacterial blight. XofabF1 A strain that has undergone one-time gene recombination.
[0039] (4) Obtaining the secondary recombinant strain Using the suicide vector pK18mobsacB sacB The gene was used as a negative selection marker (strains carrying this gene cannot grow on sucrose-containing media). Single colonies of the above-mentioned recombinant strains were picked and inoculated into 5 mL of sucrose-free NA liquid (3 g beef extract, 5 g peptone, and 1 g yeast extract per liter) and cultured overnight at 30°C with shaking at 200 rpm. The cultured bacterial solution was serially diluted with sterile water and spread on NA plates containing 15% sucrose and incubated upside down at 30°C for 3-4 days.
[0040] Single colonies grown on the plates were streaked onto NA+Kan resistant plates and NA-non-Kan resistant plates, respectively. Colonies that grew normally on NA-non-Kan resistant plates but not on NA+Kan resistant plates were selected for PCR verification using primer pairs pK18-XofabF1 P1 and pK18-XofabF1 P14. Strains whose PCR amplification product band size matched the expectation were selected for sequencing verification. Strains with correct amplified fragments and loss of kanamycin resistance were identified as *D. oryzae*, the pathogen causing bacterial blight of rice. XofabF1 Gene knockout strains (i.e., Δ) XofabF1 (mutant strain) (see) Figure 1 ).
[0041] Example 2: Determination of Pathogenicity of Rice The pathogenicity of rice was tested using the leaf-cutting method. The rice variety was Nipponbare (…). Rice L.ssp. Japanese cultivar Nipponbare ). Different treatment groups of strains ( Xoo strain (wild-type rice bacterial blight pathogen strain) and △ XofabF1 (Mutant strain) Incubated overnight at 200 rpm and 30°C with shaking for 24 h, then OD was measured. 600 =0.5, use sterilized scissors to dip into the bacterial solution, cut about 1.0-2.0 cm from the tip of the second and third leaves of rice and mark it; after inoculation, the rice is kept moist and cultured in a greenhouse at 28℃ and observed daily. After 14 days, 30 leaves are randomly selected to measure the length of the lesions.
[0042] Among them, vaccination △ XofabF1 mutant strains and Xoo Actual footage of rice leaf infection by the strain is shown below. Figure 2 As shown, the results indicated that rice leaves inoculated with the wild-type pathogen developed distinct white / yellowish-brown necrotic spots, which were long, spread rapidly, and caused severe leaf wilting; inoculation with △ XofabF1 The mutant strain produced short and shallow necrotic spots on rice leaves, and the leaves remained largely healthy. These results indicate that the knockout strain... XofabF1 After the gene was modified, the ability of pathogens to infect rice and cause diseases was greatly reduced.
[0043] In addition, vaccination △ XofabF1 mutant strains and Xoo Analysis of statistical results of rice lesion length of strains as follows: Figure 3 As shown, the results indicate that Xoo The average lesion length of the strain group was much greater than Δ XofabF1 The mutant strain group showed statistically significant differences. These results indicate that knockout... XofabF1The gene can significantly reduce the pathogenicity of rice bacterial blight, and the effect is stable and reliable.
[0044] Example 3 Growth Curve Measurement Different treatment groups of strains ( Xoo strains and △ XofabF1 The mutant strain was cultured overnight at 30°C with shaking at 200 rpm for 48 h. 10 µL of the above bacterial culture was then transferred to 1 mL of liquid NA medium (3 g beef extract, 5 g peptone, 1 g yeast extract per liter), thoroughly mixed, and 300 µL was transferred to a microbial growth assay plate. The plate was shaken at 200 rpm and incubated at 30°C. The OD of the bacterial culture was recorded every 2 h. 600 Values are used to plot growth curves.
[0045] The results are as follows Figure 4 As shown, the results indicate that Xoo The growth curve of this strain rises rapidly with a high peak, indicating a fast reproduction rate; △ XofabF1 The mutant strain exhibited a slower growth curve with a lower peak value, and its reproduction rate was significantly reduced. These results indicate that knockout... XofabF1 After genetic modification, the ability of pathogens to reproduce and spread within rice plants weakens, making it difficult for them to quickly form a "bacterial abundance advantage," thus hindering the spread of the disease.
[0046] Example 4: Detection of extracellular polysaccharides Different treatment groups of strains ( Xoo strains and △ XofabF1 (Mutant strain) Incubate overnight at 30°C with shaking at 200 rpm for 48 hours, then adjust to OD. 600 =2.0, inoculate at a ratio of 2% into 50mL NA containing 4% glucose, and incubate at 30℃ and 200rpm for 5 days. Add 4 times the volume of pre-cooled anhydrous ethanol to precipitate the extracellular polysaccharide while stirring. Then place in a -20℃ refrigerator overnight. Then remove the flocculent precipitate (if necessary, centrifuge at low speed) and place in a 60℃ oven to dry thoroughly before weighing.
[0047] The results are as follows Figure 5 As shown in the figure, the results show that △ XofabF1 The extracellular polysaccharide production of the mutant strain was much lower than that of the mutant strain. Xoo Strains. The above results indicate that knocking out... XofabF1 After genetic modification, pathogens find it difficult to colonize the surface / body of rice and are easily cleared by the rice's defense system, thus preventing continuous infection.
[0048] Example 5: Motion Performance Measurement Different treatment groups of strains ( Xoo strains and △ XofabF1 (Mutant strain) Incubate overnight at 30°C with shaking at 200 rpm for 48 hours, then adjust to OD.600 =2.0, use a pipette to take 2 μL of bacterial suspension and inoculate it onto an NA medium plate containing 0.5% agarose; incubate at 30℃ for 3 days, observe and record the colony size.
[0049] The results are as follows Figure 6 As shown, the results indicate that Xoo The strain has large, loose colonies and is highly motile; △ XofabF1 The mutant strain produced small, dense colonies that were almost impossible to move independently. These results indicate that knocking out... XofabF1 After gene mutation, the pathogen's ability to move and spread within rice tissues is reduced; it can only survive near the infection point and cannot spread to the entire rice plant.
[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. The role of XoFabF1 protein and its encoding gene in regulating rice bacterial blight (… Xanthomonas oryzae pv. oryzae Applications in virulence: The amino acid sequence of the XoFabF1 protein is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the XoFabF1 protein is shown in SEQ ID NO.
2.
3. The application according to claim 1 or 2, characterized in that, Application of reducing XoFabF1 protein activity or knocking out or down the gene encoding XoFabF1 protein in reducing the virulence, growth rate, extracellular polysaccharide synthesis, or motility of rice bacterial blight pathogen.
4. Rice bacterial blight pathogen Δ XofabF1 The mutant strain or its suspension or lyophilized powder is characterized by, Rice bacterial blight pathogen Δ XofabF1 Mutant strains were created by knocking down or eliminating the pathogen of rice bacterial blight. XofabF1 Gene acquisition.
5. The rice bacterial blight pathogen Δ as described in claim 4 XofabF1 A method for constructing mutant strains, characterized in that, The construction method includes: using a suicide vector as a vector, primer amplification to obtain... XofabF1 Upstream and downstream gene fragments are digested with two enzymes to... XofabF1 The upstream and downstream gene fragments were inserted into an enzyme digestion vector to construct the pK18-XofabF1 homologous recombination vector, which was then transformed into a wild-type strain of *Bacillus oryzae* var. *rice* and cultured to obtain *Bacillus oryzae* Δ. XofabF1 Mutant strain.
6. The construction method according to claim 5, characterized in that, The suicidal vector is pK18mobsacB.
7. XofabF1 The application of genes as targets in screening drugs to control rice bacterial blight pathogens: the drugs can reduce the expression level of the XoFabF1 protein encoding gene of rice bacterial blight pathogens or inhibit the activity of XoFabF1 protein.