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[Delta]Tlrgt2 trichoderma engineering bacteria with high yield of peptaibols and construction method and application thereof

A construction method and technology of antimicrobial peptides, applied in the field of bioengineering, can solve the problems of unsatisfactory application of peptaibols antimicrobial peptides, high price, etc.

Active Publication Date: 2019-03-01
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alamethicin from Sigma aldrich is the only commercialized peptaibols antimicrobial peptide, but the price is too high to satisfy the application of peptaibols antimicrobial peptides in the fields of agriculture and medicine

Method used

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  • [Delta]Tlrgt2 trichoderma engineering bacteria with high yield of peptaibols and construction method and application thereof
  • [Delta]Tlrgt2 trichoderma engineering bacteria with high yield of peptaibols and construction method and application thereof
  • [Delta]Tlrgt2 trichoderma engineering bacteria with high yield of peptaibols and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The construction of the ΔTlrgt2 knockout vector is as follows:

[0075] 1. Design and synthesis of primers

[0076] According to the upstream and downstream 2500bp positions of the Trichoderma SMF2Tlrgt2 gene, primers were designed for cloning the upstream homology arm sequence and downstream homology arm sequence of the gene; primers were designed according to the hygromycin resistance gene hph for cloning from the pUCATPH plasmid Hygromycin resistance gene hph sequence; according to the N terminal of the upstream homology arm, the downstream homology arm C terminal design primers for the amplification of ΔTlrgt2 knockout vector; the nucleotide sequence of the Tlrgt2 gene is as SEQ ID No Shown in .1, the amino acid sequence encoded by the Tlrgt2 gene is shown in SEQ ID No.2, the nucleotide sequence of the upstream homology arm sequence of the Tlrgt2 gene is shown in SEQ ID No.3, and the downstream homology arm of the Tlrgt2 gene The nucleotide sequence of the sequence...

Embodiment 2

[0104] The construction of ΔTlrgt2 Trichoderma engineering bacteria, the steps are as follows:

[0105] 1. Preparation of Trichoderma SMF2 Protoplasts

[0106] 1) Prepare spores at a concentration of 5×10 7 ~5×10 8 Individual / mL Trichoderma SMF2 spore suspension, take 1mL and inoculate in 50mL mycelial growth medium, culture at 28°C and 160rpm for 13.5h to obtain mycelial culture;

[0107] 2) Centrifuge the mycelium culture obtained in step 1) at 4000 rpm for 5 min, wash the precipitate with 0.7M NaCl solution three times, 25 mL each time, to obtain mycelium;

[0108] 3) Add 6 mL of cell wall lysing enzyme solution to the mycelia obtained in step 3), resuspend, and enzymatically hydrolyze at 30°C and 60 rpm for 2.5 to 3 hours; observe through a microscope, and when a large number of free protoplasts appear, use a G2 funnel to filter , add 15mL pre-cooled 0.7M NaCl solution to the filtrate, centrifuge at 3800rpm at 4°C for 5min to collect protoplasts, wash the collected prot...

Embodiment 3

[0134] The peptaibols antimicrobial peptide yield analysis of ΔTlrgt2 Trichoderma engineering bacteria, the steps are as follows:

[0135] 1. Fermentation and cultivation of ΔTlrgt2 Trichoderma engineering bacteria

[0136] The constructed ΔTlrgt2 Trichoderma engineering bacteria and Trichoderma SMF2 strains were respectively inoculated in PDB medium, and cultured at 28° C. and 180 rpm for 10 days to obtain a strain culture solution, and each strain was set in 3 replicates;

[0137] The above PDB medium components are as follows: Peel the potatoes, weigh 200g, cut into pieces, add appropriate amount of water and boil for 30min, filter through 4 layers of gauze, add 20g of glucose, add distilled water to make up to 1L;

[0138] 2. HPLC detection of peptaibols antimicrobial peptides

[0139] Centrifuge the strain culture solution obtained in step 1 at 10,000rpm for 20min to obtain a crude extract, and use the crude extract with Agela C 18 The extraction and concentration of th...

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Abstract

The invention discloses [delta]Tlrgt2 trichoderma engineering bacteria with high yield of peptaibols and a construction method and application thereof. A Tlrgt2 knockout vector is constructed by upstream and downstream homologous arm sequences of a Tlrgt2 gene and a hygromycin resistance gene hph sequence, the [delta]Tlrgt2 trichoderma engineering bacteria is prepared by a gene knockout method, aTlrgt2 gene is knocked out, and the peptaibols production capacity of the bacteria can be effectively improved. The stable passage of the constructed [delta]Tlrgt2 trichoderma engineering bacteria canbe achieved, the yield of the trichoderma peptaibols can be significantly increased, and the production of the peptaibols by the [delta]Tlrgt2 trichoderma engineering bacteria is about 12 times higher than that of an original strain. The application of the trichoderma peptaibols in medicine and agriculture is facilitated.

Description

technical field [0001] The invention relates to a ΔTlrgt2 trichoderma engineering bacterium with high production of peptaibols antibacterial peptides, a construction method and application thereof, and belongs to the technical field of bioengineering. Background technique [0002] Trichoderma spp. is an important fungal resource, which widely exists in soil, decayed wood, rhizosphere and phyllosphere. Trichoderma is not only an important enzyme production strain in industry, but also an important bio-control factor and plant growth-promoting factor in agricultural production. The biocontrol mechanism of Trichoderma mainly includes the following aspects: (1) Hyperparasitic effect: Trichoderma can recognize the host fungus, not only attach and wrap around the host mycelium, but also produce a variety of extracellular enzymes to dissolve the host cell wall and penetrate Mycelium absorbs nutrients; (2) Antibiotic effect: Trichoderma can produce a variety of secondary metabolite...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C07K14/37C12R1/885
CPCC07K14/37C12N15/80
Inventor 张玉忠陈秀兰周燕蓉宋晓妍张熙颖解彬彬苏海楠
Owner SHANDONG UNIV
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