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High-toxicity metarhizium anisopliae strain and construction method thereof

A technology of Metarhizium anisopliae and high toxicity, which is applied in the field of genetic engineering, can solve the problems of low control efficiency, slow insecticidal effect, and restriction of wide application, and achieves the effects of improving sporulation time, increasing spore production and reducing cost.

Pending Publication Date: 2022-03-18
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Metarhizium anisopliae, like other entomopathogenic fungi, has the problems of slow insecticide and low control efficiency, which seriously restricts its wide application.

Method used

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  • High-toxicity metarhizium anisopliae strain and construction method thereof
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  • High-toxicity metarhizium anisopliae strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of PK2-PB-MaAL-L / R recombinant plasmid

[0032] 1. Primer design

[0033] PCR primers were designed according to the genome sequence of Metarhizium anisopliae (WT genome) to amplify the upper and lower homology arm fragments of the MaAL gene (GeneID: 19251869). Source arm amplification primers, base sequences are shown in Table 1. Described Metarhizium anisopliae (CQMa102 bacterial strain) is provided by Chongqing University Insecticidal Fungus Biological Pesticide Engineering Technology Research Center, this bacterial strain is preserved in China General Microorganisms Collection Center, and its preservation number is CGMCCNo.0877, has applied for authorization, patent authorization announcement No. CN 1216144C.

[0034] Table 1 Amplification primers for upper and lower homology arm fragments of Metarhizium anisopliae MaAL gene

[0035]

[0036] 2. PCR amplification of upper and lower homology arm fragments of MaAL gene

[0037] PCR amplif...

Embodiment 2

[0096] Example 2 Construction of Reply Vector (PK2-sur-MaAL-egfp)

[0097] 1. Primer design and target fragment PCR amplification

[0098] Design the reply primers MaAL_CF / MaAL_CR (18bp and 19bp carrier adapter recognition sequences are added to the 5′ of the upstream and downstream primers respectively) base sequence is shown in Table 4, and the 2949bp upstream of the start codon ATG of the Metarhizium anisopliae MaAL gene is amplified to the stop codon All sequences of the sub-TGA. The target fragment was amplified and restored using the WT genome as a template, and the amplified target fragment was detected on agarose gel (1%), and the target fragment was purified and recovered using a purification kit. The ORF gene sequence of the MaAL gene's own promoter and coding region is shown in SEQ ID NO.23.

[0099] Table 4 Metarhizium anisopliae MaAL gene promoter and open reading frame amplification primers

[0100]

[0101] PCR amplification system (25μL):

[0102] ...

Embodiment 3

[0116] Transformation of embodiment 3 Agrobacterium and co-cultivation with Metarhizium anisopliae

[0117] 1. Chemical transformation of Agrobacterium

[0118] Place 5 μl of the PK2-PB-MaAL-L / R plasmid in 50 μL of Agrobacterium competent (purchased by Biotech Biological Company), bathe in ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, and treat it in a water bath at 37°C for 5 minutes, then place it on ice Place on top for 5 minutes and then add 600 μl LB liquid culture based on 28 ° C, 220 rpm shaker culture for 3 hours, take 5 μ L spread on LK medium, colony culture, positive transformant was verified by colony PCR.

[0119] The PK2-sur-MaAL-egfp vector plasmid was also transformed into Agrobacterium in the same way, and positive transformants were confirmed by PCR.

[0120] 2. Co-cultivation of Agrobacterium and Metarhizium anisopliae

[0121] 1) Inoculate the successfully verified Agrobacterium-positive colonies into 20 mL of LK liquid medium (LB liquid mediu...

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Abstract

The invention provides a high-toxicity metarhizium anisopliae engineering strain and a construction method thereof, the engineering strain is an engineering strain of metarhizium anisopliae CQMa102 obtained by knocking out a MaAL gene encoding a main allergen Asp f2 protein, and the MaAL is a newly discovered virulence gene. The knockout MaAL gene is replaced by a recombinant gene composed of an upstream homologous arm of the gene, a marker gene and a downstream homologous arm of the gene. A locust drip bioassay test result shows that the median lethal time is 1.53 days earlier than that of a wild strain (WT) and is shortened by 24.75%, and the sporulation quantity of the high-toxicity metarhizium anisopliae engineering strain is increased by 44% than that of the wild strain, so that the production cost of the strain is effectively reduced. The invention is very important for further digging the molecular mechanism of insect pathogenic fungus infection pathopoiesis and enhancing the availability of the strain, and provides a novel insecticidal fungicide.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the improvement and obtaining of a high-virulence Metarhizium anisopliae engineering strain by means of a genetic engineering method. Background technique [0002] Entomopathogenic fungi are the most important group of insecticidal microorganisms. They have the advantages of being specific to hosts, safe for humans and animals, and friendly to the environment. of biopesticides. [0003] In 1873, LeConte put forward a proposal about using microorganisms to control insects (Leconte, 1873, The American Naturalist., 7(12):710-722). Metarhizium anisopliae is the earliest entomopathogenic fungus used to control pests. The research and practice on its pest control has been more than 100 years. So far, there are more than 100 registered Metarhizium anisopliae insecticide products at home and abroad, in Africa, Australia, Countries such as Brazil and China have b...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/31C12N15/80A01N63/30A01P7/04C12R1/645
CPCC07K14/37C12N15/80A01N63/30Y02A50/30
Inventor 彭国雄夏玉先金玉梅汪杰
Owner CHONGQING UNIV