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CN-122011140-A - Method for rapidly identifying drug resistance of bakanae disease germ to fenugreek

CN122011140ACN 122011140 ACN122011140 ACN 122011140ACN-122011140-A

Abstract

The invention discloses identification of a FfMyosin nucleotide point mutation of a related gene FfMyosin-5 of bakanae disease germ and application thereof in monitoring resistance of a bactericide cyanomycoester, and particularly discloses a molecular detection method for identifying that 422 or 218 amino acid mutation of the FfMyosin-5 gene of bakanae disease germ has resistance to cyanomycoester and a special primer thereof. The molecular detection method provided by the invention has the advantages of high sensitivity, good stability and short detection period, and can be used for monitoring the frequency of resistance genes of field rice bakanae germ to the bactericide fenvinasse and the occurrence and development of resistance.

Inventors

  • ZHANG CAN
  • Jing Junlu
  • GENG SHAN
  • LIU XILI
  • LIU ZHANYUN
  • ZHANG QINGHUA
  • Chang Zhengjie
  • HUANG ZHONGQIAO

Assignees

  • 中国农业大学

Dates

Publication Date
20260512
Application Date
20251203

Claims (10)

  1. 1. The protein related to resistance of bakanae disease germ to cyanogen mycoester is protein shown in the following 1) or 2): 1) A protein consisting of the amino acid residue sequence of SEQ ID NO. 21 of the sequence Listing; 2) A protein which is derived from the SEQ ID NO. 21 and is related to the resistance function of the fenugreek through the substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid residue sequence of the SEQ ID NO. 21 in the sequence table; In SEQ ID NO. 21, X=E or A at position 422 from the amino terminus, and X=K or N at position 218 from the amino terminus.
  2. 2. The gene encoding a protein related to resistance to cyhalothrin of bakanae disease of rice as defined in claim 1.
  3. 3. The coding gene according to claim 2, wherein the genomic DNA sequence of the coding gene is as set forth in 1), 2) or 3): 1) The nucleotide sequence of SEQ ID NO. 22 in the sequence table; 2) A DNA molecule which is capable of recombining with the genomic DNA sequence of 1) under stringent conditions and which encodes a protein having a functional association with resistance to cyanurate; 3) A DNA molecule which has more than 90 percent of homology with the nucleotide sequence of SEQ ID NO. 22 in the sequence table and codes a protein related to the resistance function of the cyanogen allyl ester; Wherein, in SEQ ID NO. 22, nucleotide m=a or c at 1320 from the 5' end. In addition, from the 5' -end, at nucleotide 709 s=g or c.
  4. 4. An expression cassette, recombinant expression vector or transgenic recombinant bacterium comprising the coding gene of claim 2 or 3.
  5. 5. The application of the bakanae disease germ myoglobin 5 of rice or the coding gene thereof in the identification of the drug resistance of the bakanae disease of the rice.
  6. 6. The use according to claim 5, wherein the resistance of bakanae disease germ to cyanomycoester is increased by detecting whether the 422 th amino acid from the N-terminus of bakanae disease germ myosin 5 is changed from glutamic acid to alanine and the 218 th amino acid from the N-terminus is changed from lysine to asparagine, wherein bakanae disease germ myosin 5 is the protein of claim 1.
  7. 7. The use according to claim 5, wherein the resistance to cyanurate is increased by detecting a nucleotide 1320 from the 5 'end of the genomic DNA encoding myoglobin 5 of bakanae disease of rice, if the 1320 th nucleotide is c, and a further nucleotide 709 from the 5' end is g, if the 709 th nucleotide is c, the resistance to cyanurate is also increased, and the encoding gene of myoglobin 5 of bakanae disease of rice is the encoding gene as set forth in claim 2 or 3.
  8. 8. A primer group for detecting or assisting in detecting whether a FfMyosin-5 gene in bakanae disease of rice has mutation sites or not is composed of a DNA molecule shown as SEQ ID NO. 11 and 15 in a sequence table, and a DNA molecule shown as SEQ ID NO. 16 and 20 in the sequence table.
  9. 9. A method for detecting or assisting in detecting whether a FfMyosin-5 gene in bakanae disease of rice has a mutation site or not comprises the following steps: taking genome DNA of bakanae disease germ to be detected as a template, carrying out PCR amplification by using a primer pair consisting of a fragment shown in SEQ ID NO. 11 and a fragment shown in SEQ ID NO. 15, and if the primer pair consisting of the fragment shown in SEQ ID NO. 11 and the fragment shown in SEQ ID NO. 15 can amplify a 474bp band, then FfMyosin-5 genes in bakanae disease germ to be detected exist or mutation sites exist in candidates; In addition, the genome DNA of the rice bakanae germ to be detected is used as a template at the mutation site, a primer pair consisting of a fragment shown in SEQ ID NO. 16 and a fragment shown in SEQ ID NO. 20 is used for carrying out PCR amplification, and if a 388bp band can be amplified by the primer pair consisting of the fragment shown in SEQ ID NO. 16 and the fragment shown in SEQ ID NO. 20, the FfMyosin-5 gene in the rice bakanae germ to be detected exists or the mutation site is candidate to exist; Preferably, in the PCR amplification, the annealing temperature of SEQ ID NO. 11 and SEQ ID NO. 15 is 58 ℃. Another mutation, SEQ ID NO. 16 and SEQ ID NO. 20, annealing temperature is 64 ℃; Preferably, the mutation site refers to mutation from 1320 st nucleotide from 5' end to c of FfMyosin th gene in rice bakanae disease germ, thereby causing 422 st amino acid from N end of rice bakanae disease germ myosin 5 to be changed from glutamic acid to alanine; The other mutation is characterized in that the mutation site refers to that the 709 th nucleotide of FfMyosin-5 gene in the bakanae disease germ is mutated into c from the 5' end, thereby leading the 218 th amino acid of the bakanae disease germ myosin 5 from the N end to be changed into asparagine from lysine; Preferably, the mutant bakanae disease germ myoglobin 5 is shown as SEQ ID NO. 21, wherein 422 th amino acid from the N end of the SEQ ID NO. 21 is alanine, and FfMyosin-5 coding gene in the mutant bakanae disease germ is shown as SEQ ID NO. 22, wherein 1320 th nucleotide from the 5' end of the SEQ ID NO. 22 is c; the other characteristic is that the mutant rice bakanae germ myoglobin 5 is shown as SEQ ID NO. 21, wherein the 218 th amino acid from the N end of the SEQ ID NO. 21 is asparagine, the FfMyosin-5 coding gene in the mutant rice bakanae germ is shown as SEQ ID NO. 22, and the 709 th nucleotide from the 5' end of the SEQ ID NO. 22 is c.
  10. 10. The primer group shown in claim 8 or the application of the method shown in claim 9 in identifying the drug resistance of bakanae disease germ, wherein the drug resistance is the drug resistance of bakanae disease germ to fenvinasse; Preferably, the rice bakanae germ with the mutation site has or is candidate to have drug resistance.

Description

Method for rapidly identifying drug resistance of bakanae disease germ to fenugreek Technical Field The invention belongs to the technical field of molecular biology. Relates to identification of nucleotide point mutation on a related gene FfMyosin-5 of bakanae disease germ myoglobin and application thereof in detection of resistance of bactericides cyanomycoesters, in particular to a molecular detection method for rapidly identifying the nucleotide point mutation of the FfMyosin-5 gene of bakanae disease germ and resistance of the bakanae disease germ to cyanomycoesters and a special primer. Background Bakanae disease (rice bakanae disease) of rice, also known as hiking disease, white stem disease, was first discovered in japan in 1828. Bakanae disease is a worldwide disease of rice that has been distributed throughout the world in rice planting areas including asia, africa, america and european countries. In China, bakanae disease of rice occurs in various rice areas of China. Bakanae disease of rice can occur in the period from seedling stage to heading stage, and symptoms can occur in the period from 2 to 4 leaves of the rice seedling. Typical symptoms of bakanae disease of rice are the fading and yellowing of leaves of the ill plant, abnormal elongation and growth of leaves caused by gibberellic acid production, maldevelopment of root system of the ill plant, shortened root length, white to light pink mold production at the base of stem of the ill plant, namely conidium and conidium peduncles of the strain. In addition, plant internodes are obviously elongated, the joints are bent, a plurality of aerial roots are generated on the joints, the tillering capability is weakened, and the like are also the onset symptoms of bakanae disease of rice. The Honda generally shows symptoms within 20-30 days after transplanting, and if the rice is too heavy in disease, the rice dies before heading. Colorless conidia are produced on leaf sheaths of dead rice plants. The disease plants with light disease can heading, but the rice ears are short and the grains are few. The registered medicines for preventing and controlling rice seedlings are 69 products (comprising single agents and compound agents), and the effective components of the products mainly comprise prochloraz, carbendazim, thiophanate-methyl, tebuconazole, cyhalothrin, fludioxonil, metalaxyl, hymexazol, fluazinam, ethylicin and the like. However, with the unreasonable and frequent use of these fungicides, many phytopathogens have developed serious resistance to these fungicides. Therefore, each large agricultural chemical company in the world is continuously striving to develop a novel bactericide which has a brand new action mechanism and has no orthogonal mutual drug resistance with the existing bactericides on the market, and is used for preventing and controlling rice bakanae disease and controlling drug resistance. The fenugreek is a bactericide which is developed by Jiangsu base of the national south pesticide creation center in 1998 and has complete independent intellectual property rights. Is mainly used for preventing and treating diseases caused by fusarium infection such as wheat scab, rice bakanae disease, cotton wilt and the like. The cyanogen mycoester chemical structure belongs to cyanoacrylate bactericides, and the action mechanism research shows that the cyanogen mycoester targets Myosin coded by fusarium Myosin-5 gene and is combined in a pocket of a Myosin combined slit to prevent the Myosin from being combined with F-actin (cytoskeleton) so as to further play a bacteriostatic activity. The committee on action on resistance to bactericides (Fungicide Resistance Action Committee, FRAC) classifies the cyanomycoesters as cyanoacrylate bactericides based on their chemical structure. The cyanogen alkene fungus esters are registered in China in 2007, and are mainly used for preventing and treating diseases caused by Fusarium. The current committee on action on resistance to bactericides (FRAC) sets its inherent risk of resistance to high levels, which require resistance risk management during use. The research laboratory monitors in the field to obtain the rice bakanae-resistant strain which shows high-level resistance to the fenvinpoconazole, and the survival suitability of the resistant strain is higher, which indicates that the field resistance risk of the rice bakanae-resistant strain to the fenvinpoconazole is medium to high and the like. Therefore, when using the cyanogen alkene fungus ester to prevent and cure the bakanae disease of the rice, the resistance monitoring needs to be enhanced, and the resistance occurrence condition of the cyanogen alkene fungus ester needs to be timely early-warned, so that the scientific use of the cyanogen alkene fungus ester is guided, the generation of the drug resistance is delayed, and the service life of the bactericide is prolonged. Conventional methods for detecting resistance of bactericides incl