A method for rapid identification of resistance of rice bakanae pathogen to cyanocobalamin

Abstract

The present invention discloses the identification of nucleotide point mutations in the myosin 5-related gene FfMyosin‑5 of rice seedling pathogen and its application in monitoring resistance to the fungicide cyanothiocarb, and specifically discloses a molecular detection method and special primers for identifying the resistance of rice seedling pathogen FfMyosin‑5 gene 218 amino acid mutation to cyanothiocarb. The molecular detection method provided by the present invention has high sensitivity, good stability and short detection cycle, and can be used for high-throughput monitoring of the frequency of resistance genes of rice seedling pathogen to the fungicide cyanothiocarb in the field and the occurrence and development of resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, relates to the identification of nucleotide point mutations on the myosin 5-related gene FfMyosin-5 of rice seedling pathogen and its application in the detection of fungicide cyanothiocarb, and specifically relates to a molecular detection method and special primers for rapid identification of nucleotide point mutations in the FfMyosin-5 gene of rice seedling pathogen and its resistance to cyanothiocarb. Background Art

[0002] Fusarium fujikuroi, a fungus of rice seedling disease, belongs to the phylum Ascomycota, class Hyphomycete, order Tuberculariales, genus Fusarium Link ex Fr. It is an important pathogen in rice production. Rice seedling disease is a seed-borne disease that can occur during the entire growth and development period of rice. When the seeds are infected, the germination rate can be sharply reduced. The diseased plants will show leggy growth, yellow leaves, abnormal root development, and often die before transplanting at the 2-4 leaf stage. The peak period of symptoms is also 10-30 days after transplanting. The internodes of the diseased plants are elongated, the nodes are exposed to the outside of the leaf sheath, and the tillering ability is weakened. Dark brown stripes can be seen on the stems inside the leaf sheath, and white mycelium like spider silk can be observed, and the plants will then slowly wither. Plants with mild symptoms will ear early, and the ears are small and not solid. The disease occurs during the heading period, and the ears of grain will turn brown after infection and cannot bear fruit. When encountering continuous rainy weather, black ascocarps can be observed on the surface of the diseased plants. There are 69 products (including single agents and compound agents) registered in my country for the prevention and control of rice seedling disease. The active ingredients of the products are mainly prochloraz, carbendazim, methyl thiophanate, tebuconazole, cyanocobalamin, fludioxonil, oxadone, fluazinam and ethoxyconazole. However, with the irrational and frequent use of these fungicides, many plant pathogens have developed serious resistance to the above fungicides. Therefore, major international agrochemical companies have been committed to developing new fungicides with a new mechanism of action and no positive cross-resistance with existing fungicides on the market, which are used for the prevention and control of rice seedling disease and resistance management.

[0003] Cyanostrobin is a fungicide developed by the Jiangsu Base of the National Southern Pesticide Creation Center in 1998 with completely independent intellectual property rights. It is mainly used to prevent and treat diseases caused by Fusarium infection, such as wheat fusarium head blight, rice seedling disease, and cotton wilt. Cyanostrobin belongs to the cyanoacrylate fungicide in terms of chemical structure. Studies on its mechanism of action have shown that cyanostrobin targets myosin encoded by the Myosin-5 gene of Fusarium and binds to the pocket of the myosin binding cleft, preventing myosin from binding to F-actin (cytoskeleton), thereby exerting antibacterial activity. The Fungicide Resistance Action Committee (FRAC) classifies cyanostrobin as a cyanoacrylate fungicide based on its chemical structure. Cyanostrobin obtained pesticide registration in my country in 2007 and is mainly used to prevent and treat diseases caused by Fusarium. Currently, the Fungicide Resistance Action Committee (FRAC) has classified its inherent resistance risk as high, and resistance risk management is required during its use. Our laboratory obtained rice seedling resistance strains that showed high levels of resistance to cyanocobalamin in field monitoring, and the survival fitness of the resistant strains was high, indicating that the field resistance risk of rice seedling disease pathogens to cyanocobalamin was medium to high. Therefore, when using cyanocobalamin to control rice seedling disease, it is necessary to strengthen resistance monitoring and provide timely warnings for the occurrence of cyanocobalamin resistance, so as to guide the scientific use of cyanocobalamin, delay the development of its resistance, and extend the service life of the fungicide.

[0004] Conventional methods for detecting fungicide resistance include: mycelium growth rate method, mycelium dry weight determination method, spore germination determination method and agar spreading method. However, these methods require the isolation and purification of pathogens first, and then the pathogens are inoculated on drug-containing culture media or on living plants or tissues treated with fungicides, and the results can only be investigated after a certain period of time. Using conventional methods, when the frequency of resistant strains in the field is greater than 1%, they can be detected (for example, if there is a 95% probability of detecting resistant strains with a frequency of 1%, the sample size required for detection needs to be greater than 300), so it has the disadvantages of low sensitivity, large workload, and long test cycle.

[0005] With the rapid development of molecular biology technology and the continuous expansion of its application scope, restriction enzyme PCR and allele-specific PCR molecular technology have begun to be used in the detection of pathogen resistance. Compared with traditional detection methods, it not only saves detection time and improves work efficiency, but also enhances the sensitivity of detection. The detection frequency is 10 -5 -10 -4 , is more suitable for detecting low-frequency resistance genes and is also regarded as an ideal method for early diagnosis of resistance in the field. Therefore, molecular detection technology will play an increasingly important role in the sustainable management system of diseases. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a rice seedling pathogen resistance-related protein to cyanostrobin and its encoding gene, as well as the rice seedling pathogen resistance to cyanostrobin screening marker, the application of the protein in the rice seedling pathogen resistance to cyanostrobin.

[0007] In order to achieve the object of the present invention, the technical solution of the present invention is as follows:

[0008] One object of the present invention is to provide a protein related to resistance to cyanothiocarb in rice seedling pathogen, which is a protein shown in 1) or 2) below:

[0009] 1) A protein consisting of the amino acid residue sequence of SEQ ID NO: 8 in the sequence listing;

[0010] 2) A protein derived from SEQ ID NO: 8 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added and the amino acid residue sequence is related to the cyanobacterium-resistance function.

[0011] The above SEQ ID NO: 8 is as follows:

[0012]

[0013]

[0014] Wherein, X (218th position from the amino terminal) = K or R.

[0015] Among them, in the rice seedling pathogen cyanothiocarb resistance-related protein, if the 218th amino acid is lysine, the pathogen is sensitive to cyanothiocarb, and if the 218th amino acid from the N-terminus is mutated from lysine to arginine, the rice seedling pathogen is resistant to cyanothiocarb.

[0016] In one embodiment of the present invention, the sequence of the cyanothiocarb resistance-associated protein (myosin 5) of the rice seedling pathogen is as shown in SEQ ID NO:8. When the 218th amino acid from the N-terminus in SEQ ID NO:8 is lysine (X=K), the rice seedling pathogen is sensitive to cyanothiocarb, and when the 218th amino acid from the N-terminus of SEQ ID NO:8 is arginine (X=R), the rice seedling pathogen is resistant to cyanothiocarb.

[0017] The present invention also provides a gene encoding a protein related to cyanocobalamin resistance.

[0018] The genomic nucleotide sequence of the coding gene is shown in 1), 2) or 3) below:

[0019] 1) the nucleotide sequence of SEQ ID NO: 9 in the sequence listing;

[0020] 2) a DNA molecule that can be recombined with the DNA sequence described in 1) under stringent conditions and encodes a protein related to cyanocobalamin resistance function;

[0021] 3) A DNA molecule having more than 90% homology with the nucleotide sequence of SEQ ID NO: 9 in the sequence listing and encoding a protein related to cyanocobalamin resistance function.

[0022] The above SEQ ID NO:9 is as follows:

[0023]

[0024]

[0025]

[0026] In SEQ ID NO: 9, the 708th nucleotide from the 5' end is r = a or g.

[0027] In one embodiment of the present invention, the coding gene is as shown in SEQ ID NO: 9, and the 708th nucleotide from the 5' end of SEQ ID NO: 9 is A (r=a), which is a genotype sensitive to cyanocobalamin.

[0028] When the 708th nucleotide from the 5' end is mutated to G (the 708th nucleotide from the 5' end in SEQ ID NO:9 is r=g), it is a genotype showing resistance to cyanocobalamin (the 708th nucleotide from the 5' end in its target gene is mutated to G).

[0029] The expression cassette, recombinant expression vector or transgenic recombinant bacteria containing the above-mentioned coding gene also fall within the protection scope of the present invention.

[0030] Another object of the present invention is to provide the use of myosin 5 of rice seedling pathogen or its encoding gene in identifying the resistance of rice seedling pathogen to cyanothiocarb.

[0031] The use of myosin 5 of rice seedling pathogen or its encoding gene as a target for screening pesticides against drug-resistant rice seedling pathogen also falls within the protection scope of the present invention.

[0032] In one embodiment of the present invention, by detecting whether the 218th amino acid from the N-terminus of myosin 5 of rice seedling pathogen is changed from lysine to arginine, if the 218th amino acid is changed from lysine to arginine, its resistance to cyanocobalamin is improved; wherein, myosin 5 of rice seedling pathogen is the protein shown in SEQ ID NO: 8 in the sequence table.

[0033] By detecting that the 708th nucleotide from the 5' end of the coding gene of myosin 5 of rice seedling pathogen is G, the resistance to cyanocobalamin is improved; the coding gene of myosin 5 of rice seedling pathogen is the coding gene shown in SEQ ID NO:9.

[0034] Another object of the present invention is to provide a primer set for detecting or assisting in detecting whether there is a mutation site in the FfMyosin-5 gene in rice bakanae pathogen, comprising the DNA molecules described by SEQ ID NO: 6 and SEQ ID NO: 7 in the sequence listing.

[0035] The application of the primer set in identifying the drug resistance of rice bakanae pathogen also belongs to the protection scope of the present invention; the drug resistance refers to the resistance of rice bakanae pathogen to cyanostrobin.

[0036] The present invention also provides a method for detecting or assisting in detecting whether there is a mutation site in the FfMyosin-5 gene in rice bakanae pathogen, comprising the following steps:

[0037] Using the genomic DNA of the rice bakanae pathogen to be tested as a template, PCR amplification is performed using the primer pair shown in SEQ ID NO: 6 and SEQ ID NO: 7. If SEQ ID NO: 6 and SEQ ID NO: 7 can amplify a 741 bp band, the FfMyosin-5 gene in the rice bakanae pathogen to be tested has or is a candidate for a mutation site.

[0038] In the PCR amplification, the annealing temperature of the primer pair SEQ ID NO: 6 and SEQ ID NO: 7 is 61°C.

[0039] In the method, the mutation site refers to the 708th nucleotide from the 5' end of the FfMyosin-5 gene in the rice seedling pathogen being G, thereby causing the 218th amino acid from the N end of the myosin 5 of the rice seedling pathogen to mutate from lysine to arginine.

[0040] In the method, the mutated myosin-5 of rice seedling pathogen is as shown in SEQ ID NO:8, wherein the 218th amino acid from the N-terminus of SEQ ID NO:8 is arginine (X=R), and the mutated FfMyosin-5 gene in rice seedling pathogen is as shown in SEQ ID NO:9, wherein the 708th nucleotide from the 5' end of SEQ ID NO:9 is G.

[0041] The present invention also provides application of the method in identifying the drug resistance of rice bakanae pathogen; the drug resistance refers to the drug resistance of rice bakanae pathogen to cyanostrobin.

[0042] The rice bakanae pathogen having the mutation site has or is a candidate for having drug resistance.

[0043] Another object of the present invention is to provide a mutation site of the FfMyosin-5 gene or protein in rice bakanae pathogen.

[0044] The mutation site of the FfMyosin-5 gene in the rice bakanae pathogen provided by the present invention is that the 708th nucleotide from the 5' end of the genomic DNA sequence of the FfMyosin-5 gene of the rice bakanae pathogen mutates to G, and the nucleotide mutation exists alone.

[0045] The mutation site of myosin 5 in rice bakanae pathogen provided by the present invention is that the 218th amino acid from the N-terminus of myosin 5 in rice bakanae pathogen is arginine.

[0046] The mutation site refers to the genotype that is sensitive to cyanothiocarb, where the 708th position of the FfMyosin-5 gene in the rice seedling pathogen from the 5' end is mutated to G. As a result, the 218th amino acid of the myosin 5 from the N end of the rice seedling pathogen is changed from lysine to arginine.

[0047] The application of any of the above mutation sites in identifying the resistance of rice bakanae pathogen to pesticides also falls within the protection scope of the present invention; in the above application, the rice bakanae pathogen with the mutation site has or is a candidate for having drug resistance. The drug resistance refers to the resistance of rice bakanae pathogen to cyanothiocarb.

[0048] The molecular detection method provided by the present invention has high sensitivity, good stability and short detection cycle, and can be used to monitor the frequency of resistance genes of rice bakanae disease to the fungicide cyanocobalamin in the field and the occurrence and development of resistance in a high-throughput manner, so as to achieve early warning of resistant diseases, guide timely adjustment of the prevention and control strategy of rice bakanae disease, promote scientific use of drugs, delay the occurrence and development of drug resistance, and play an important role in the sustainable management system of diseases.

[0049] The method provided by the present invention for detecting the point mutation of the resistance of rice bakanae pathogen to cyanocobalamin can perform high-sensitivity, simple and rapid molecular detection on the resistant strain, timely understand the dynamics of drug resistance, and is of great significance for formulating reasonable disease management programs and effectively controlling the development of drug-resistant populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Amino acid sequence alignment of sensitive and resistant strains of rice bakanae pathogen (K218R)A. Gene pattern of rice bakanae FfMyosin-5 and the location of the K218R point mutation on the gene; B. Amino acid sequence alignment of FfMyosin-5 protein in rice bakanae pathogen-sensitive and resistant strains.

[0051] LNPJX2-1, SCMS-7 and AHLJ-2 were sensitive strains; HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 were resistant strains carrying the K218R point mutation.

[0052] Figure 2 Figure 4 is the binding mode of cyanocobalamin and rice seedling-sensitive FfMyosin-5 protein. A. Amino acids that interact with cyanocobalamin and FfMyosin-5 protein; B. The binding posture and binding energy of cyanocobalamin and FfMyosin-5 protein.

[0053] Figure 3 Figure 4 shows the binding mode of cyproconazole and the FfMyosin-5 protein of the rice seedling K218R mutant. A. Amino acids that interact with cyproconazole and K218R-FfMyosin-5 protein; B. The binding posture and binding energy of cyproconazole and K218R-FfMyosin-5 protein.

[0054] Figure 4 AS-PCR primers and annealing temperature screening for K218R point mutation A is K218R-F1; B is K218R-F2; C is K218R-F3; D is K218R-F4.

[0055] Figure 5 The results show that the AS-PCR detection of rice seedling pathogen resistant strains (61℃) is for the K218R point mutation; A site specificity, among which LNPJX2-1, SCMS-7 and AHLJ-2 are sensitive strains; HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 are resistant strains carrying the K218R type point mutation; B species specificity, among which HLJJX-3, HLJJX-25, HLJJX-29 are resistant strains carrying the K218R mutation site, S219P-1 and S219P-2 are resistant strains carrying the S219P mutation site, Fusarium graminearum is Fusarium graminearum, Fusariumpseudograminearum is Pseudofusarium graminearum, Rhizoctonia solani is the rice sheath blight pathogen, Pyricularia oryzae is the rice blast pathogen, and Ustilaginoidea oryzae is the rice false smut pathogen. DETAILED DESCRIPTION

[0056] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0059] The above strains are only the materials used in the examples of the present invention. In fact, when applying the screening markers of the present invention, any fungal strains obtained from commercial sources can be used.

[0060] In the text, "resistant strains" refer to the rice seedling pathogen that is resistant to the fungicide cyanocobalamin; "sensitive strains" refer to the rice seedling pathogen that is sensitive to the fungicide cyanocobalamin.

[0061] The rice seedling-killing strains used in the following examples are: strains LNPJX2-1, LNPJX2-6, LNPJX2-7, SCMS-6, SCMS-7 and AHLJ-2 are sensitive strains, and strains HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 are resistant strains.

[0062] The above strains LNPJX2-1, LNPJX2-6 and LNPJX2-7 were collected from Panjin City, Liaoning Province, China; SCMS-6 and SCMS-7 were collected from Meishan City, Sichuan Province, China; AHLJ-2 was collected from Lujiang County, Anhui Province, China; HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 were collected from Jixi City, Heilongjiang Province, China; all strains were identified as rice seedling pathogens by existing morphological and molecular biological methods. The above strains were all verified for drug resistance by colony growth assay.

[0063] Example 1: Field resistance monitoring of rice seedling pathogen cyanothiocarb

[0064] Rice Bakanae pathogens were collected from all over the country for field resistance monitoring. After monitoring, it was found that HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 collected from Jixi City, Heilongjiang Province, China were all resistant strains. LNPJX2-1, LNPJX2-6 and LNPJX2-7 collected from Panjin City, Liaoning Province, China; SCMS-6 and SCMS-7 collected from Meishan City, Sichuan Province, China; AHLJ-2 collected from Lujiang County, Anhui Province, China were all sensitive strains.

[0065] Example 2: Sensitivity of Bakanae oxyphylla to cyanothiocarb

[0066] HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 were selected from the monitored rice seedling pathogen-resistant strains; LNPJX2-1, LNPJX2-6, LNPJX2-7, SCMS-6, SCMS-7 and AHLJ-2 were selected from the rice seedling pathogen-sensitive strains.

[0067] PDA medium: 1000 g potato, 100 g glucose, 15 g agar powder, distilled water to 1 L, sterilize at 121°C for 20 min.

[0068] The experimental steps are as follows:

[0069] 1) Cyanocyanobacteryl was prepared with dimethyl sulfoxide (DMSO) as solvent at a concentration of 10 5 The sensitivity of sensitive strains of rice seedling pathogen to cyanostrobin was determined by diluting the cyanostrobin stock solution with DMSO into working solutions of corresponding concentrations. The final concentrations of cyanostrobin in the drug-containing medium were 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.50 μg / mL and 1 μg / mL, respectively. The sensitivity of resistant strains of rice seedling pathogen to cyanostrobin was determined by diluting the cyanostrobin stock solution with DMSO into working solutions of corresponding concentrations. The final concentrations of cyanostrobin in the drug-containing medium were 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL.

[0070] 2) Cool the sterilized PDA medium to 45°C, add 100 μL of the corresponding drug working solution to 100 mL of PDA medium and mix well, pour the drug-containing medium into a culture dish with a diameter of 9 cm, use no drug as the blank control, and set up 3 technical replicates for each concentration of drug-containing medium.

[0071] 3) The rice seedling pathogen was cultured on a PDA plate in the dark for 4 days, and a bacterial cake with a diameter of 5 mm was punched along the edge of the colony using a borer. The mycelium side was inoculated downward on the drug-containing and control medium plates prepared in step 2), and the plates were placed in an incubator at 25° C. in the dark. The colony diameter was measured after 4 days.

[0072] 4) The colony diameter was measured by the cross method, and the inhibition rate of each concentration of the agent on the mycelial growth of the test strain was calculated based on the colony diameter. The inhibition rate was then converted into a probability value (Y), and the concentration of the agent was converted into a logarithmic value with a base of 10 (X). The virulence regression equation Y = a + bX and the correlation coefficient R of the tested rice seedling spore strain were respectively obtained using the DPS data processing software. 2 and effectively inhibited the concentration of EC 50 value and calculate the resistance multiple.

[0073] Resistance multiple = EC of resistant strain 50 EC / susceptible strains 50

[0074] The results are shown in Table 1:

[0075] Table 1. Sensitivity of tested rice seedling strains to cyanocbiconazole

[0076]

[0077]

[0078] The sensitivity of rice seedling pathogen to cyanothiocarb was tested and the results showed that the average EC 50 was 0.253 μg / mL, while the average EC of the resistant strains HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 50 The average EC of sensitive strains was 26.305 μg / mL, which was significantly higher than the average EC of sensitive strains. 50 As shown in Table 2. The resistance multiple is 103.97.

[0079] Example 3: Discovery of mutation sites of FfMyosin-5 gene and myosin 5 in rice bakanae pathogen

[0080] 1. Strain culture

[0081] 1. Strains: The sensitive strains are LNPJX2-1, LNPJX2-6, LNPJX2-7, SCMS-6, SCMS-7 and AHLJ-2; the resistant strains are HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49.

[0082] 2. Methods:

[0083] Strain culture: PDB medium (1000 g potato, 100 g glucose, distilled water to 1 L, 121°C, sterilized for 20 min for use), cultured rice seedling pathogen at 25°C. The pre-cultured rice seedling pathogen sensitive and resistant strains were inoculated on PDB medium, cultured in the dark at 25°C for 4 days, collected hyphae, frozen in liquid nitrogen and stored at -80°C for genomic DNA extraction.

[0084] 2. Extract genomic DNA of sensitive and resistant strains of rice seedling pathogen respectively:

[0085] 1) Take an appropriate amount of mycelium frozen in liquid nitrogen, grind it into powder with a mortar and pestle, and place it in a 2 mL centrifuge tube;

[0086] 2) Add 800 μL of 3% CTAB solution (preheated at 65°C) to the centrifuge tube, then place in a 65°C water bath for 1 hour, mixing by inversion for 15 minutes;

[0087] 3) Add equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) to the centrifuge tube, mix well, and centrifuge at 12000g for 10 min at room temperature;

[0088] 4) Pipette 600 μL of supernatant into a new 2 mL sterile centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), place on a 3D shaker at 75 rpm, room temperature, for 10 min;

[0089] 5) Centrifuge at 12000g for 10 min at room temperature, take 400 μL of supernatant into a new centrifuge tube, add 0.6 times the volume of isopropanol, and place in a -20°C refrigerator for 30 min;

[0090] 6) Centrifuge at 12000g, 4°C, 10 min, remove the supernatant and keep the precipitate, which is DNA;

[0091] 7) Add 800 μL of 75% ethanol to the centrifuge tube and tap the tube wall to wash the DNA;

[0092] 8) 7500g, 4°C, 5 min, centrifuge to remove supernatant, and dry under reduced pressure to remove alcohol;

[0093] 9) Add 30-50 μL ddH2O to the tube containing DNA and measure the DNA concentration and purity.

[0094] Store at -20℃ until used.

[0095] 3. Amplification of the actin gene FfMyosin-5 of sensitive strains of rice bakanae pathogen

[0096] Primers FfMyosin-5-F (5'-TTTGAGACAACCAAGAAGAAG-3', SEQ ID NO: 1) and FfMyosin-5-R (5'-TGCATAGCATTCTTCCTGGC-3', SEQ ID NO: 2) were used to amplify the full length of the gene encoding Myosin-5 of rice seedling pathogen.

[0097] PCR reaction system (50 μL reaction system): FastPfu Fly DNA Ploymerase 1 μL, dNTPs 4 μL, FastPfu Fly Buffer 10 μL, FfMyosin-5-F 1 μL (10 μM), FfMyosin-5-R 1 μL (10 μM), genomic DNA 100 ng (1 μL), ddH2O to 50 μL. PCR reaction program is as follows: 94.0℃ for 3 min; then 94.0℃ for 30 s, 55℃ for 30 s, 72.0℃ for 120 s, 35 cycles; finally 72.0℃ for 10 min; stored at 12℃.

[0098] The PCR product was sequenced. The results showed that the myosin 5FfMyosin-5 gene of the sensitive strain of rice bakanae pathogen was 3799 bp in length, as shown in SEQ ID NO:9, which contained a 109 bp intron. After removing the intron, the cDNA sequence was 3690 bp in length, and the 708th nucleotide from the 5' end of SEQ ID NO:9 was A (r=a), encoding the amino acid sequence shown in SEQ ID NO:8, and the 218th amino acid at the N-terminus of SEQ ID NO:8 was lysine (X=K).

[0099] The above SEQ ID NO: 8 is as follows:

[0100]

[0101] Wherein, X (218th position from the amino terminal) = K or R.

[0102] The above SEQ ID NO:9 is as follows:

[0103]

[0104]

[0105] In SEQ ID NO: 9, the 708th nucleotide from the 5' end is r = a or g.

[0106] 4. Sequence Alignment

[0107] The full length of the Myosin-5 gene of the resistant strains HLJJX-29, HLJJX-43, HLJJX-49, HLJJX-37, HLJJX-3, HLJJX-35, HLJJX-25 and the sensitive strains HLJJX-30, HLJJX-24 and HLJJX-46 were sequenced. The sequence was analyzed and compared by DANMAN software, and the K218R resistance mutation type was found in the cyanobacterium-resistant strains. The specific results are as follows:

[0108] In the sensitive strain, the 708th nucleotide from the 5' end of the genomic DNA sequence of the FfMyosin-5 gene is A (the 708th nucleotide from the 5' end of SEQ ID NO:9 is r=a), and the 218th amino acid from the N-terminus of the corresponding FfMyosin-5 protein is lysine (Lys) (the 218th amino acid from the N-terminus of SEQ ID NO:8 is lysine (X=K)), while the 708th nucleotide from the 5' end of the genomic sequence of the Myosin-5 gene in the resistant strains HLJJX-3, HLJJX-25, HLJJX-29, HLJJX-35, HLJJX-37, HLJJX-43 and HLJJX-49 is G (the 708th nucleotide from the 5' end of SEQ ID NO:9 is r=g), and the 218th amino acid from the N-terminus of the corresponding FfMyosin-5 protein is arginine (Arg) (the 218th amino acid from the N-terminus of SEQ ID NO:8 is lysine (X=K)). The 218th amino acid from the N-terminus of NO:8 is arginine (X=R) ( Figure 1 ).

[0109] The results showed that the type of mutation in the FfMyosin-5 protein of the resistant strain was a point mutation from A to G at the 708th position from the 5' end of the genome (the 218th nucleotide from the 5' end of SEQ ID NO:9 was G (r=g)). Analysis of the peak line graph of the sequencing showed an obvious single peak, and the change in the 708th base also led to the mutation of lysine at position 218 to arginine, that is, the mutation type in which the 218th amino acid at the N-terminus of SEQ ID NO:8 was arginine (X=R).

[0110] Example 4: Verification of resistance-related mutation sites in rice bakanae pathogen

[0111] 1. Sequence Alignment

[0112] The amino acid sequence of the FfMyosin-5 protein of rice seedling pathogen was obtained by sequencing in our laboratory. The crystal structure in the protein database (Protein data bank, PDB) was searched by the Protein BLAST tool of NCBI. In the search results, the sequence consistency of the crystal structure 6UI4 and the Myosin-5 protein reached 97.40%, with a high homology. Therefore, the 6UI4 structure can be used as a template for constructing the Myosin-5 structure.

[0113] 2. Homology Modeling

[0114] Based on the template protein three-dimensional structure file and sequence alignment file, the structural similarity was inferred from the sequence similarity, and the Modeller v9.19 program was used to perform homology modeling on the FfMyosin-5 protein to obtain a reasonable three-dimensional structural model of the target protein, and the protein model was optimized by molecular mechanics.

[0115] The FfMyosin-5 protein model was structurally optimized using the Amber7 force field. The optimization process was carried out in two steps: first, 2000 steps of steepest descent optimization were performed, and then 2000 steps of conjugate gradient method were used to further optimize the structure. The final result was used as the model for subsequent analysis.

[0116] 3. Molecular Docking

[0117] The constructed FfMyosin-5 protein and FfMyosin-5-K218R protein structures were subjected to molecular mechanics optimization and used as receptors for molecular docking. Molecular docking was implemented using the AutoDock4.2.6 software package. The structure of the substrate cyanobacterium methyl was obtained, hydrogenated, and the structure was optimized using the MOPAC program to calculate the PM3 atomic charge. Finally, AutodockTools1.5.6 was used to process the structures of the ligand and receptor respectively, and the docking box was wrapped around the active site. The number of grid points in the XYZ direction was set to 25.6×25.6×25.6, and the grid spacing was Amino acids 218 and 219 were used as the center of the docking box, the number of dockings was set to 200, and the remaining parameters used the default values.

[0118] IV. Results Analysis

[0119] 1) Interaction between cyproconazole and FfMyosin-5 protein and its mutant K218R

[0120] In order to analyze the driving force of cyproconazole binding to Myosin-5 protein and mutant K218R, the interaction mode of cyproconazole-Myosin-5 protein was analyzed. Cyproconazole binds to a hydrophobic pocket composed of 12 amino acid residues, including Leu215, Leu216, Glu217, Lys218, Ser219, Met377, Phe421, Glu422, Cys425, Lys539, Asp542 and Ala579, among which Lys218 and Lys539 bind to cyproconazole by hydrogen bonds ( Figure 2 A). It can stabilize the binding between cyanocobalamin and FfMyosin-5, and the binding energy is -7.19kcal / moL ( Figure 2 B). However, after the K218R point mutation of FfMyosin-5 protein, cyanocobalamin binds to the hydrophobic pocket composed of 10 amino acid residues, including Arg218, Ala374, Met377, Glu422, Asp538, Lys539, Lys541, Asp542, Gln543 and Leu545, among which Lys541 and Gln543 form hydrogen bonds with cyanocobalamin ( Figure 3 A). Not only the number of amino acid residues involved in molecular interactions is reduced, but also after the K218R mutation, due to the influence of steric hindrance, the posture of cyanothiocarb is changed, and the amino acids involved in the hydrogen bond interaction are changed, resulting in its binding energy with the target being reduced to -6.46kcal / moL ( Figure 3 B), thereby making the FfMyosin-5-K218R (the 218th amino acid at the N-terminus of SEQ ID NO: 8 is arginine (X=R)) mutant strain show resistance.

[0121] V. Conclusion

[0122] The molecular docking method was used to study the reason why mutant K218R produces resistance. The molecular docking results showed that cyproconazole can bind to the pocket of the myosin 5 binding cleft, preventing myosin from binding to F-actin (cytoskeleton), thereby exerting antibacterial activity. The mutation of Lys218 to Arg218 can change the binding mode of cyproconazole with the target, and the number of surrounding amino acid residues involved in the interaction is reduced, and the binding ability of cyproconazole with the active center is reduced, so that the FfMyosin-5-K218R mutant strain exhibits drug resistance.

[0123] Example 5: Method for detecting mutation sites in rice bakanae pathogen

[0124] 1. Primer Design

[0125] The strain was the strain used in Example 1, and the primers were as shown in Table 3. Allele Specific-PCR primers were designed according to the sequence of the mutant FfMyosin-5 gene, and the penultimate base of the 3'-end of the forward primer was consistent with the mutated base. In order to increase the specificity of the primer, mismatched bases A, T, G and C were introduced into the last base of the 3'-end of the forward primer respectively; the reverse primers were as shown in Table 2.

[0126] Table 2. Primers used in AS-PCR detection of cyanothiocarb resistance in rice seedling pathogens

[0127]

[0128]

[0129] In this method, the second to last base mutant at the 3'-end of the upstream primer is consistent, and the last base is also changed, and amplification is performed in combination with different annealing temperatures to improve primer specificity.

[0130] The PCR reaction system is as follows:

[0131] 20μL PCR system: Easy Taq 0.5μL, dNTPs 2μL, Easy Taq Buffer 2μL, K218R-F (1 / 2 / 3 / 4) 0.8μL (10μM), K218R-R 0.8μL (10μM), genomic DNA 100ng (1μL), ddH2O to 20μL.

[0132] The PCR reaction conditions were as follows: 94°C for 3 min first; then 94°C for 30 s, 55-65°C for 30 s, 72°C for 45 s, 35 cycles; finally 72°C for 10 min; and stored at 12°C.

[0133] The results of PCR product amplification showed that K218R-F2, F3 and F4 could amplify a 741bp band from the resistant strain at an annealing temperature of 61°C, but could not amplify a band from the sensitive strain ( Figure 5 B / C / D), considering the primer specificity, K218R-F4 / K218R-R was selected. The above results show that the primer pair K218R-F4 / K218R-R can detect the K218R resistance site respectively, so the primer pair K218R-F4 / K218R-R (61°C) was selected as the AS-PCR primer to detect the above mutation site. The amplification product of primers K218R-F4 / K218R-R is nucleotides 635-1376 in SEQ ID NO:9.

[0134] Detection of mutation sites

[0135] The genomic DNA of the rice seedling pathogen to be tested is used as a template, and the primer pair K218R-F4 / K218R-R is used for PCR amplification, and then agarose gel electrophoresis is performed. If a fragment can be amplified from the genomic DNA of the strain, the strain is determined to be a resistant strain. If a fragment cannot be amplified, the tested rice seedling pathogen is determined to be a sensitive strain.

[0136] The PCR amplification system and reaction conditions were consistent with those in the experiment, and the annealing temperature of the primer pair K218R-F4 / K218R-R was 61°C.

[0137] The strains to be tested for primer pair K218R-F4 / K218R-R are: resistant strains HLJJX-29, HLJJX-43, HLJJX-49, HLJJX-37, HLJJX-3, HLJJX-35 and HLJJX-25; sensitive strains HLJJX-30, HLJJX-24 and HLJJX-46.

[0138] The results showed that the primer pair K218R-F4 / K218R-R could only amplify a 741 bp fragment from the resistant strain at an annealing temperature of 61°C, but could not amplify a band from the sensitive strain ( Figure 4 D).

[0139] Therefore, the primer pair K218R-F4 / K218R-R can be used to effectively detect the resistant strains of rice seedling pathogen to cyanocobalamin under the annealing temperature of 61°C.

Claims

1. A protein related to resistance of Bakanae oxysporum to cyanocobalamin, which is a protein consisting of the amino acid residue sequence of SEQ ID NO: 8 in the sequence table; wherein, In SEQ ID NO: 8, position 218 from the amino terminus is arginine.

2. The gene encoding the protein related to the resistance of Bakanae oxyphylla to cyanocobalamin according to claim 1.

3. The coding gene according to claim 2, characterized in that: The genomic DNA sequence of the encoding gene is shown in SEQ ID NO: 9 in the sequence listing, wherein the 708th nucleotide from the 5' end of SEQ ID NO: 9 is G.

4. An expression cassette, a recombinant expression vector or a transgenic recombinant bacterium containing the coding gene according to claim 2 or 3.

5. Use of myosin 5 of rice seedling pathogen or its encoding gene in identifying resistance to rice seedling pathogen or as a target for screening pesticides against resistant rice seedling pathogen; In the identification of the resistance of rice seedling disease, by detecting whether the 218th amino acid from the N-terminus of myosin 5 of rice seedling disease pathogen changes from lysine to arginine, the above change will increase the resistance of rice seedling disease pathogen to cyanothiocarb; wherein, The mutated myosin 5 of rice bakanae pathogen is the protein according to claim 1; In the identification of the resistance to rice bakanae disease, the 708th nucleotide from the 5' end of the genomic DNA encoding myosin 5 of rice bakanae disease pathogen is detected to be A. If the 708th nucleotide mutates to G, the resistance to cyanocobalamin is improved; the mutated coding gene of myosin 5 of rice bakanae disease pathogen is the coding gene shown in claim 3.

6. A primer set for detecting or assisting in detecting whether there is a mutation site in the gene encoding myosin 5 of rice seedling pathogen in rice seedling pathogen, consisting of the DNA molecule shown in SEQ ID NO: 6 and the DNA molecule shown in SEQ ID NO: 7 in the sequence listing.

7. A method for detecting or assisting in detecting whether a mutation site exists in a gene encoding myosin 5 of Bakanae oxysporum f. oryzae, comprising the following steps: Using the genomic DNA of the rice bakanae pathogen to be tested as a template, PCR amplification is performed using a primer pair consisting of the fragment shown in SEQ ID NO: 6 and the fragment shown in SEQ ID NO:

7. If the primer pair consisting of the fragment shown in SEQ ID NO: 6 and the fragment shown in SEQ ID NO: 7 can amplify a 741 bp band, then the gene encoding myosin 5 of rice bakanae pathogen to be tested has or is a candidate for a mutation site; The mutation site refers to the mutation of the 708th nucleotide from the 5' end of the myosin 5 gene of the rice seedling pathogen in the rice seedling pathogen to G, thereby causing the 218th amino acid from the N-terminus of the myosin 5 of the rice seedling pathogen to change from lysine to arginine; The mutated myosin 5 of Bakanae oryzae is shown in SEQ ID NO: 8, wherein: The 218th amino acid from the N-terminus of SEQ ID NO: 8 is arginine; the gene encoding myosin 5 of bakanae oxyphylla in the mutated bakanae oxyphylla is shown in SEQ ID NO: 9, wherein the 708th nucleotide from the 5' end of SEQ ID NO: 9 is G.

8. The method according to claim 7, characterized in that In the PCR amplification, the annealing temperature of SEQ ID NO: 6 and SEQ ID NO: 7 is 61°C.

9. Use of the method according to claim 7 or 8 or the primer set according to claim 6 in identifying the drug resistance of rice bakanae pathogen; the drug resistance is the resistance of rice bakanae pathogen to cyanostrobin.

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