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CN-121991974-A - High-toxicity metarhizium anisopliae, and construction method and application thereof

CN121991974ACN 121991974 ACN121991974 ACN 121991974ACN-121991974-A

Abstract

The invention discloses a high-toxicity metarhizium anisopliae and a construction method and application thereof. The MaTri gene can regulate and control the virulence and/or spore yield of metarhizium anisopliae, the construction method of metarhizium anisopliae with high virulence uses metarhizium anisopliae CQMa421 as an original strain, obtains the full-length sequence of an open reading frame of MaTri through PCR amplification, clones the sequence into an expression vector pK2-PgpdM-pB to construct a recombinant super-expression vector, adopts an agrobacterium-mediated transformation method to introduce the recombinant vector into the metarhizium anisopliae CQMa421, and obtains MaTri super-expression strain OE-MaTri through resistance screening and molecular verification. The half-death time of the overexpression strain to the sogatella furcifera is shortened by approximately 1 d compared with that of a wild strain, the spore production capacity is obviously enhanced, and the resistance to damp heat, ultraviolet and chemical substances is kept stable. The super-expression strain constructed by the invention has strong pathogenicity and good environmental adaptability, can effectively prevent and control the sogatella furcifera, and provides high-quality strain resources and technical support for the development of fungal biopesticides.

Inventors

  • XIE JIAQIN
  • CAO YINGCHUN
  • WANG LEI
  • XIA YUXIAN

Assignees

  • 重庆大学

Dates

Publication Date
20260508
Application Date
20260212

Claims (10)

  1. The application of the MaTri gene in regulating and controlling the virulence and/or spore yield of metarhizium anisopliae is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO.1 or the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2.
  2. 2. The use of claim 1, wherein modulating fungal virulence comprises positively modulating fungal virulence or negatively modulating fungal virulence.
  3. 3. A method for cultivating high-virulence metarhizium anisopliae engineering fungi is characterized by comprising the step of overexpressing MaTri genes in receptor fungi to obtain the high-virulence engineering fungi, wherein the nucleotide sequence of the genes is shown as SEQ ID NO.1 or the amino acid sequence of protein coded by the genes is shown as SEQ ID NO. 2.
  4. 4. A high-virulence metarhizium anisopliae engineering fungus is characterized in that MaTri genes are overexpressed in the engineering fungus, the nucleotide sequence of the genes is shown as SEQ ID NO.1, or the amino acid sequence of protein encoded by the genes is shown as SEQ ID NO. 2.
  5. 5. The engineered fungus of claim 4, wherein said overexpression comprises the steps of: (1) The MaTri gene fragment is connected with vector pK2-PgpdM-pB which is cut by XbaI and XhoI through recombinase to obtain recombinant super-expression vector pK2-PgpdM-MaTri; (2) Transferring the recombinant overexpression vector pK2-PgpdM-MaTri into an agrobacterium competent cell to obtain an agrobacterium strain containing the recombinant overexpression vector; (3) Co-culturing the agrobacterium strain containing the recombinant overexpression vector with spore suspension of engineering fungi, and screening to obtain MaTri overexpression strain.
  6. 6. The engineered fungus of claim 4, wherein the engineered fungus is metarhizium anisopliae.
  7. 7. An insecticide, characterized in that the active ingredient comprises the engineering fungus of any one of claims 4 to 6.
  8. 8. Use of the engineering fungus according to any one of claims 4 to 6 for controlling pests.
  9. 9. The use of claim 8, wherein the pest comprises sogatella furcifera.
  10. 10. Use of an engineered fungus according to any one of claims 4 to 6 for the production of spores.

Description

High-toxicity metarhizium anisopliae, and construction method and application thereof Technical Field The invention relates to the technical field of microbial genetic engineering, in particular to a high-toxicity metarhizium anisopliae, a construction method and application thereof. Background The sogatella furcifera is one of main pests of rice, and seriously threatens the safe production of the rice by piercing and sucking phloem juice of the rice and transmitting viruses. The long-term use of chemical pesticides leads to the problems of enhanced drug resistance of pests, environmental pollution and the like, and the entomopathogenic fungi serving as biological pesticides have the advantages of environmental safety, uneasiness in producing resistance of hosts and the like, and become an important direction for green prevention and control of pests. Metarhizium anisopliae (Metarhizium anisopliae) is a broad-spectrum entomopathogenic fungus, and the strain CQMa421 of the metarhizium anisopliae has good application potential in preventing and controlling rice pests, but the wild strain has the defects of low insecticidal speed, limited pathogenicity and the like, so that the large-scale application of the metarhizium anisopliae is limited. At present, the function of MaTri genes in metarhizium anisopliae is not clear, and the research on improving the pathogenicity of metarhizium anisopliae by a gene overexpression technology has not been reported. Disclosure of Invention The invention aims to provide high-toxicity metarhizium anisopliae, a construction method and application thereof, and the pathogenicity of metarhizium anisopliae to sogatella furcifera is enhanced by over-expression MaTri genes, and meanwhile, the environmental adaptability of the metarhizium anisopliae is maintained, so that high-efficiency strain and technical support are provided for green prevention and control of rice pests. In view of the above, maTri gene is applied to regulating and controlling the virulence and/or spore yield of metarhizium anisopliae, the nucleotide sequence of the gene is shown as SEQ ID NO.1, or the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2. Further, the modulating fungal virulence includes positively modulating fungal virulence or negatively modulating fungal virulence. Preferably, the metarhizium anisopliae is metarhizium anisopliae CQMa421. The second object of the invention is to provide a method for cultivating high-virulence metarhizium anisopliae engineering fungi, which comprises the step of overexpressing MaTri genes in receptor fungi to obtain the high-virulence engineering fungi, wherein the nucleotide sequence of the genes is shown as SEQ ID NO.1, or the amino acid sequence of protein coded by the genes is shown as SEQ ID NO. 2. Preferably, the metarhizium anisopliae engineering fungus is metarhizium anisopliae CQMa421. The invention further aims to provide a high-virulence metarhizium anisopliae engineering fungus, maTri genes are overexpressed in the engineering fungus, the nucleotide sequence of the genes is shown as SEQ ID NO.1, or the amino acid sequence of protein coded by the genes is shown as SEQ ID NO. 2. Preferably, the engineering fungus is metarhizium anisopliae CQMa421; Further, the overexpression comprises the following steps: (1) The MaTri gene fragment is connected with vector pK2-PgpdM-pB which is cut by XbaI and XhoI through recombinase to obtain recombinant super-expression vector pK2-PgpdM-MaTri; (2) Transferring the recombinant overexpression vector pK2-PgpdM-MaTri into an agrobacterium competent cell to obtain an agrobacterium strain containing the recombinant overexpression vector; (3) Co-culturing the agrobacterium strain containing the recombinant overexpression vector with spore suspension of engineering fungi, and screening to obtain MaTri overexpression strain; Preferably, the engineering fungus is metarhizium anisopliae CQMa421; preferably, the agrobacterium competent cells are agrobacterium AGL1 competent cells; further, the method further comprises the step of cloning MaTri genes before obtaining the recombinant overexpression vector pK2-PgpdM-MaTri in the step 1), wherein the genomic DNA of metarhizium anisopliae CQMa421 is used as a template, and the specific primers are used for PCR amplification to obtain the full-length sequence SEQ ID NO.1 of the open reading frame of the MaTri genes, and the specific primer sequences are as follows: MaTri-F:ctctccatatacacacacgcaaatctagaATGGAGACGGACTCATTTAA; MaTri-R:ctcgcccttgctcaccatactagtctcgagATGTTTATAAGCACCGACTC; preferably, the PCR amplification is performed under reaction conditions of 98℃for 2 min, 98℃for 10 s,58℃for 30 s,72℃for 30 s cycles, and 72℃for 4 min. Further, the step 1) of the recombinant enzyme connection further comprises the steps of transforming escherichia coli DH5 alpha, colony PCR verification and sequencing identification; Further, after the recombinant overexpressio