Fusarium pseudograminearum GPI anchoring protein and application of coding gene thereof

Abstract

The invention belongs to the technical field of agricultural disease control, and discloses a fusarium pseudograminearum GPI anchoring protein and an application of a coding gene of the fusarium pseudograminearum GPI anchoring protein. The fusarium pseudograminearum FpPer1 gene is knocked out, so that the mycelial growth rate, conidium yield and pathogenicity of the fusarium pseudograminearum FpPer1 gene are remarkably inhibited, and meanwhile, the gene knockout strain is more sensitive to bactericides such as tebuconazole. The gene can be used as a novel target for designing a pesticide synergist; or a small molecule compound, polypeptide or nucleic acid medicine is designed based on the FpPer1 gene or the FpPer1 protein coded by the FpPer1 gene, and is compounded with the existing bactericide (such as tebuconazole) to improve the pesticide effect. A new target is provided for prevention and control of wheat stem rot, efficient prevention and control are achieved by interfering a pathogen protein post-translational modification approach, the toxin pollution risk is reduced, and wide agricultural application prospects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural disease control technology, specifically relating to the application of a Fusarium pseudocarpa GPI anchoring protein and its encoding gene. Background Technology

[0002] Wheat stem rot ( Fusarium Crown rot is a major soil-borne disease in wheat production, and its main pathogens include: Fusarium pseudobulb (…). Fusarium pseudograminearum Fusarium graminearum ( ), Fusarium graminearum ) and yellow Fusarium ( Fusarium culmorum Since the first reported case of Fusarium graminearum causing disease in my country in 2012, this disease has posed a continuous threat to wheat yield and quality, and there is an urgent need to develop effective control methods.

[0003] Chemical pesticides are a conventional means of pest and disease control, characterized by their high efficiency in killing insects and treating diseases. Currently, the main fungicides used to control Fusarium species such as *Fusarium graminearum* include tebuconazole, difenoconazole, flutriafol, and fludioxonil. Triadimefon and tebuconazole, as triazole fungicides, primarily work by inhibiting the biosynthesis of ergosterol (a key component of fungal cell membranes), thereby disrupting cell membrane function and controlling the pathogen. However, this class of agents has the following drawbacks: Limited target: Current research only confirms the ergosterol pathway as a target for tebuconazole, but pathogens may develop resistance through other metabolic pathways; Ecological risks: Broad-spectrum fungicides can easily disrupt the balance of soil microbial communities; Diminishing efficacy: Long-term use leads to increased pathogen resistance.

[0004] Given the limitations of existing triazole fungicides targeting the ergosterol synthesis pathway in the control of wheat stem rot, and the increasing risk of resistance to Fusarium graminearum, this invention reveals the gene encoding the GPI-anchored protein. FpPer1 Its crucial role in pathogen growth, sporulation, and drug sensitivity is proposed to be... FpPer1 Technical solutions for using genes or their encoded proteins as targets for novel pesticides. Summary of the Invention

[0005] This invention has discovered that *Fusarium pseudograss* FpPer1 Genes play a crucial regulatory role in the growth, development, and pathogenicity of pathogens: knockout FpPer1 The gene significantly inhibits mycelial growth and spore formation, and significantly increases sensitivity to agents such as tebuconazole, suggesting that FpPer1 may participate in drug resistance regulation independently of the ergosterol pathway. Therefore, FpPer1 Genes and their encoded proteins can serve as novel, specific pesticide targets, providing a new strategy to address the problems of single-target fungicides and increased resistance in existing fungicides. Specifically, this invention provides the following technical solution.

[0006] Firstly, the present application provides a nucleic acid sequence encoding a GPI anchor protein of Fusarium pseudograminearum, which is any one of the following 1) or 2): 1) the nucleic acid sequence shown as SEQ ID NO. 1; 2) the nucleic acid sequence having more than 50% homology compared with SEQ ID NO. 1.

[0007] The nucleic acid sequence can be a DNA sequence or an RNA sequence; when the nucleic acid sequence is a DNA sequence, it can be a cDNA formed by reverse transcription, a genomic DNA or an artificially synthesized DNA; when the nucleic acid sequence is an RNA sequence, it can be a miRNA, a SiRNA, a sgRNA, a shRNA or other related RNA.

[0008] The more than 50% homology preferably means more than 60% homology compared with SEQ ID NO. 1; further preferably more than 70% homology compared with SEQ ID NO. 1; further preferably more than 80% homology compared with SEQ ID NO. 1; and more preferably more than 90% homology compared with SEQ ID NO. 1.

[0009] The nucleic acid sequence shown as SEQ ID NO. 1 is a drug resistance gene of Fusarium pseudograminearum, and the drug resistance gene is FpPer1 a gene.

[0010] Secondly, the present application provides a GPI anchor protein of Fusarium pseudograminearum, which is any one of the following 1) or 2): 1) the amino acid sequence shown as SEQ ID NO. 2; 2) the amino acid sequence having more than 50% homology compared with SEQ ID NO. 2.

[0011] The more than 50% homology preferably means more than 60% homology compared with SEQ ID NO. 2; further preferably more than 70% homology compared with SEQ ID NO. 2; further preferably more than 80% homology compared with SEQ ID NO. 2; and more preferably more than 90% homology compared with SEQ ID NO. 2.

[0012] The amino acid sequence shown as SEQ ID NO. 2 is a FpPer1 protein, which is encoded by the nucleic acid sequence shown as SEQ ID NO. 1.

[0013] In a third aspect, the present application provides an application of the nucleic acid sequence or the GPI-anchored protein in the development of a pesticide target, specifically, the nucleic acid sequence or the GPI-anchored protein is used as a specific target for designing a pesticide interfering with the post-translational modification pathway of a pathogenic bacterium protein.

[0014] In a fourth aspect, the present application provides a pesticide synergist for inhibiting the transcription and / or translation of the nucleic acid sequence, so that the function thereof is inhibited or completely inactivated, thereby improving the control effect of the pesticide.

[0015] Further, for the pesticide synergist, the pesticide is a triazole fungicide or fludioxonil, and the triazole fungicide includes tebuconazole, difenconazole and epoxiconazole.

[0016] In a fifth aspect, the present application claims a biological material prepared by using the nucleic acid sequence, and the biological material includes a nucleic acid molecule, an expression cassette, an expression vector, a recombinant microorganism and a transgenic plant material, and specifically can be expressed as: 1) a nucleic acid molecule containing the nucleic acid sequence or the fragment; 2) an expression cassette containing the nucleic acid sequence or the fragment; 3) an expression vector containing the nucleic acid sequence or the fragment; 4) a recombinant microorganism containing the nucleic acid sequence or the fragment; 5) a recombinant microorganism containing 1) or 2); 6) a transgenic plant material containing the nucleic acid sequence or the fragment; 7) a transgenic plant material containing 1) or 2); The expression vector includes a viral expression vector, and the recombinant microorganism includes a recombinant fungus, a recombinant bacterium and a recombinant virus.

[0017] In a sixth aspect, the present application claims a control method of wheat foot rot, and the control method includes knocking out a GPI-anchored protein coding gene of a pathogenic bacterium causing the wheat foot rot by a homologous recombination method, and the pathogenic bacterium causing the wheat foot rot includes pseudocercosporella herpotrichoides, cereals fusarium and fusarium graminearum.

[0018] Further, in the control method, the nucleotide sequence of the GPI-anchored protein coding gene of the pseudocercosporella herpotrichoides is shown as SEQ ID NO. 1.

[0019] The present application proves, by a gene knockout experiment, that the GPI-anchored protein coding gene of the pseudocercosporella herpotrichoides (Pseudocercosporella herpotrichoides) Fusarium pseudograminearum GPI-anchored protein coding gene ‌FpPer1The deletion of the gene can significantly inhibit the mycelium growth rate, conidial production and pathogenicity of the fungus, and significantly increase the sensitivity of the fungus to triazole fungicides such as tebuconazole. Based on the above findings, the beneficial effects of the application of "a GPI-anchored protein of Fusarium pseudograminearum and the coding gene thereof" are embodied in the following aspects: 1) Development of a new pesticide target FpPer1 The gene or the FpPer1 protein encoded thereby can be used as a specific action target for designing a new biological pesticide targeting the post-translational modification pathway of pathogenic fungi. Compared with traditional ergosterol synthesis inhibitors (such as tebuconazole), this target can avoid existing drug resistance risks and provide a more precise prevention and control strategy.

[0020] 2) Application of a pesticide synergist Based on FpPer1 Small molecule compounds, polypeptides or nucleic acid drugs designed based on the gene or the FpPer1 protein encoded thereby can be used in combination with existing fungicides such as tebuconazole. By inhibiting the function of FpPer1, the sensitivity of pathogenic fungi to fungicides such as tebuconazole can be significantly enhanced, the dosage of pesticides can be reduced, and environmental residues can be reduced.

[0021] 3) Comprehensive prevention and control potential The application of this target can achieve the triple effects of pathogenic fungus growth inhibition, reproduction blockage and chemical agent synergism, providing a new solution for the comprehensive management of wheat foot rot and having a significant industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The sensitivity of different mutant strains in the mutant library of Fusarium pseudograminearum to tebuconazole was determined. Among them, WZ-8A represents the wild type Fusarium pseudograminearum strain; A9, B9, X33, X43, X47, X49, Y61 and W30 represent different mutant strains of Fusarium pseudograminearum; Tebuconazole represents PDA medium containing tebuconazole; and CK represents ordinary PDA medium.

[0023] Figure 2 The gene regulates the fungicide resistance of Fusarium pseudograminearum. A: wild type strain WZ-8A, FpPer1 Gene knockout strain Δ FpPer1 and complementation strain c Fpper1 FpPer1 ​Growth morphology of each strain on PDA medium, PDA medium containing tebuconazole, difenoconazole, epoxiconazole and fludioxonil, respectively. B: Growth inhibition rate of each strain on different PDA medium plates. Among them, CK represents PDA medium; Tebuconazole represents PDA medium containing tebuconazole; Difenoconazole represents PDA medium containing difenoconazole; Epoxiconazole represents PDA medium containing epoxiconazole; Fludioxonil represents PDA medium containing fludioxonil.

[0024] Figure 3 To FpPer1 regulate the growth rate and sporulation of F. pseudograminearum. A: Growth of wild-type strain WZ-8A, FpPer1 gene knockout strain Δ Fpper1 and complemented strain c FpPer1 B: Conidial yield of wild-type strain WZ-8A, FpPer1 gene knockout strain Δ Fpper1 and complemented strain c FpPer1 under microscopic observation (left), CFW staining of conidial septum (middle) and DIC channel and CFW fluorescence channel superimposed field observation results (right). C: Conidial yield of each strain in CMC medium (unit 1*10 6 / mL).

[0025] Figure 4 To FpPer1 regulate the pathogenicity of F. pseudograminearum to wheat and barley. A: Phenotype of wild-type strain WZ-8A, FpPer1 gene knockout strain Δ Fpper1 and complemented strain c FpPer1 after infecting wheat coleoptile sheath. B: Phenotype of wild-type strain WZ-8A, FpPer1 gene knockout strain Δ Fpper1 and complemented strain c FpPer1 after infecting barley leaves. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] The materials used in the embodiments are as follows: Test pathogenic fungi: F. pseudograminearum (Fusarium pseudograminearum ) Wild type strain WZ-8A; Mutant strains A9, B9, X33, X43, X47, X49, Y61, W30 of F. pseudograminearum.

[0028] Test agents: tebuconazole, difenoconazole, epoxiconazole and fludioxonil.

[0029] Test wheat: Zhongkang 58; Test barley: Kenpi No. 7.

[0030] Expression vector: pKNTG fungal expression vector (Wang Limin. Functional study of FpPpr1 and FpPpr5 of F. pseudograminearum pentatricopeptide repeat proteins [D]. Henan Agricultural University, 2021, 37.).

[0031] PDA medium: Take 200 g peeled potatoes, cut into small pieces, add appropriate amount of distilled water and boil until the potatoes are soft (about 20 min), filter out the residue with gauze, add 20 g glucose to the filtrate, stir well, and dilute to 1 L, sterilize at 121 ℃ for 20 min.

[0032] CMC liquid medium: 1 L: 0.5 g of magnesium sulfate heptahydrate, 1 g of ammonium nitrate, 1 g of potassium dihydrogen phosphate, 1 g of yeast extract, 10 g of carboxymethyl cellulose, sterilize at 121 ℃ for 20 min.

[0033] Example 1 This example describes the screening and identification of drug-resistant genes of F. pseudograminearum. FpPer1 F. pseudograminearum is one of the main pathogens causing wheat stem base rot. In the prevention and control of wheat stem base rot, fungicides such as tebuconazole are mainly used for chemical control. In order to identify drug-resistant related genes in F. pseudograminearum, the mutant strains in the mutant library of F. pseudograminearum preserved in the laboratory were subjected to drug resistance screening test.

[0034] Specific method: Wild type (WZ-8A strain) and mutant (A9, B9, X33, X43, X47, X49, Y61, W30 strains) of F. pseudograminearum were inoculated on PDA plates containing 0.067 ppm tebuconazole and no tebuconazole, respectively, and cultured at 25 ℃ for 3 days. The colony growth of each strain was observed.

[0035] The test results show that the mutant strain W30 of F. pseudograminearum becomes more sensitive to tebuconazole treatment

[0036] ), and the gene is determined by nested PCR to be Figure 1 (FPSE_01026). FpPer1 Example 2

[0037] This example describes the knockout FpPer1 ​The influence of genes on drug resistance in Fusarium graminearum.

[0038] To further clarify FpPer1 The influence of genes on drug resistance of Fusarium oxysporum, and the study of Fusarium oxysporum... FpPer1 Gene knockout and drug resistance identification: Gene knockout and drug resistance identification were performed using homologous recombination (referring to "Wang Limin. Functional study of FpPpr1 and FpPpr5 triangular pentapeptide repeat proteins in Fusarium oxysporum" [D]. Henan Agricultural University, 2021, 37.). FpPer1 Genes, to obtain one FpPer1 Gene knockout transformant Δ Fpper1 Furthermore, the complement strain c of this gene was constructed. FpPer1 .

[0039] Wild-type strain WZ-8A of Fusarium oxysporum, FpPer1 Gene knockout strain Δ Fpper1 and replenishment strain c FpPer1 The strains were cultured on PDA plates containing tebuconazole, difenoconazole, flutriafol, and fludioxonil, respectively, and their growth was observed. The growth inhibition rate of different strains on different drug-containing culture media plates was also calculated.

[0040]

[0041] Figure 2 for FpPer1 Gene regulation of fungicide resistance in Fusarium oxysporum. A: Wild-type strain WZ-8A, FpPer1 Gene knockout strain Δ Fpper1 and replenishment strain c FpPer1 Growth morphology of the strains on ordinary PDA medium and PDA plates containing tebuconazole, difenoconazole, flutriafol, and fludioxonil. B: Growth inhibition rate of the above strains on different drug-containing medium plates was determined. Figure 2 The results showed that, compared with wild-type strain WZ-8A and complement strain c FpPer1 In comparison, gene knockout strain Δ Fpper1 Increased sensitivity to tebuconazole, difenoconazole, flutriafol, and fludioxonil indicates that... FpPer1 Gene regulation of fungicide resistance in Fusarium graminearum.

[0042] Example 3 This embodiment describes knockout FpPer1 The influence of genes on the growth rate and conidia production of Fusarium pseudograss.

[0043] Wild-type strain WZ-8A of Fusarium oxysporum, FpPer1 Gene knockout strain ΔFpper1 and complemented strain c FpPer1 were inoculated on PDA plates respectively and incubated at 25℃ for 3 days. The results showed that the growth rate of the gene knockout strain Δ FpPer1 was slower than that of the wild type strain WZ-8A and the complemented strain c Fpper1 . Figure 3

[0044] In addition, each strain was inoculated in CMC liquid medium and incubated at 25℃, 150 rpm for 5 days. The conidial morphology was observed under microscope and the conidial yield was measured (unit: 1*10 6 / mL). The results showed that the gene knockout strain Δ Fpper1 exhibited abnormal mycelial morphology (B in FIG. 1) and the conidial yield was significantly decreased (C in FIG. 1). Figure 3 Figure 3

[0045] The results of this example showed that the gene is important for maintaining the normal life activities of F. pseudorepens. FpPer1

[0046] Example 4 This example describes the pathogenicity of the gene knockout strain Δ Fpper1 .

[0047] To further determine whether the gene regulates the pathogenicity of F. pseudorepens, the mycelial blocks of the wild type strain WZ-8A of F. pseudorepens, the gene knockout strain Δ FpPer1 and the complemented strain c FpPer1 were inoculated on barley leaves respectively. After 24 hours of dark incubation, the mycelial blocks were removed and the incubation was continued for 3 days. The infection length was measured and the photographs were taken. In addition, 2 μL of spore solution (1.0*10 7 / mL) of each strain was inoculated on the cut wheat coleoptile and incubated at 25℃ for 7 days. The lesion length was measured and the photographs were taken. Fpper1 FpPer1 The pathogenicity determination results of the wild type strain WZ-8A of F. pseudorepens, the gene knockout strain Δ

[0048] and the complemented strain c Figure 4 on the wheat coleoptile (A) and the barley leaves (B). FpPer1 The results showed that the pathogenicity of the gene knockout strain Δ Fpper1 was significantly decreased compared with the wild type strain WZ-8A and the complemented strain c FpPer1 . Therefore, the pathogenicity of F. pseudorepens was significantly decreased by knocking out the gene. Figure 4 FpPer1 Fpper1 FpPer1 ​​​​​​​​

[0049] In summary, this invention, through gene knockout experiments, confirms that the gene encoding the GPI anchoring protein of *Fusarium graminearum* is... ‌FpPer1 The absence of [a specific substance] significantly inhibits mycelial growth, reduces conidia production and pathogenicity, while significantly increasing its sensitivity to triazole fungicides and fludioxonil. Based on [a specific factor] ‌FpPer1 Functional characteristics of genes ‌FpPer1 The gene and its encoded GPI-anchored protein have important guiding significance for the development of novel pesticide targets, the application of pesticide synergists, and the integrated control of wheat diseases.

[0050] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A fungicide resistance gene of Fusarium pseudograminearum, characterized in that, The drug resistance gene is FpPer1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1. FpPer1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. A GPI-anchored protein of Fusarium pseudograminearum, characterized in that, The GPI-anchored protein is FpPer1 protein, which is encoded by the pesticide resistance gene of claim 1, and the amino acid sequence is shown as SEQ ID NO.

2.

3. Use of the drug resistance gene according to claim 1 or the GPI-anchored protein according to claim 2 in the development of a pesticide target, characterized in that, The FpPer1 Genes or GPI-anchored proteins as specific targets for the design of pesticides that interfere with the post-translational modification pathway of pathogenic proteins.

4. A pesticide synergist characterized in that, The pesticide synergist is used to inhibit the transcription and / or translation process of the pesticide resistance gene of the Fusarium pseudograminearum of claim 1, so that the function of the pesticide resistance gene is inhibited or completely inactivated, thereby improving the control effect of the pesticide.

5. The pesticide synergist according to claim 4, characterized in that, The pesticide is a triazole fungicide or fludioxonil, and the triazole fungicide includes: tebuconazole, difenoconazole, and fluquinconazole.

6. A biomaterial prepared using the anti-drug gene according to claim 1, wherein, The biological material includes: nucleic acid molecules, expression cassettes, expression vectors, recombinant microorganisms, and transgenic plant materials.

7. A method for controlling wheat take-all disease, characterized by, The control method includes: knocking out the GPI-anchored protein coding gene of the pathogenic bacteria causing wheat foot rot by the method of homologous recombination; the pathogenic bacteria causing wheat foot rot includes: Fusarium pseudograminearum, Fusarium graminearum, and Fusarium culmorum.

8. The control method according to claim 7, characterized by, The nucleotide sequence of the GPI-anchored protein coding gene of Fusarium pseudograminearum is shown as SEQ ID NO. 1.

Images