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Preparation GS115/MiAMP1 for inhibiting pear-fruit postharvest penicilliosis

A GS115, penicillium technology, applied in the direction of virus/phage, microorganism-based methods, applications, etc., can solve the problem that the biological control effect of antagonistic microorganisms cannot be achieved or approached, and achieves inhibition of penicillosis, reduction of harm, and reduction of cost effect

Inactive Publication Date: 2018-05-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological control effect of antagonistic microorganisms is still not as good as or close to that of chemical fungicides. Therefore, in order to further improve the biocontrol effect of antagonistic yeasts, researchers have carried out various attempts, including stress treatment, optimization of culture conditions, and separation and antagonism under adverse conditions. Strains and expression of exogenous genes, etc., among which, with the rapid development of molecular technology, direct transformation of antagonistic yeast with exogenous genes such as expression products that have direct antibacterial effect and can improve the stress resistance of antagonistic microorganisms has gradually become a research hotspot in the field of biological control

Method used

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  • Preparation GS115/MiAMP1 for inhibiting pear-fruit postharvest penicilliosis
  • Preparation GS115/MiAMP1 for inhibiting pear-fruit postharvest penicilliosis
  • Preparation GS115/MiAMP1 for inhibiting pear-fruit postharvest penicilliosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of recombinant expression vector

[0041] 1. Experimental materials:

[0042] Restriction endonucleases: Xho I and Not I, and T4 ligase;

[0043] Axygen's DNA gel recovery kit;

[0044] 2×Phanta Max Master Mix;

[0045] Escherichia coli competent cell DH5α;

[0046] Plasmid pPICZαA;

[0047] 2. Treatment:

[0048] (1) After the plasmid pPICZαA and the target gene containing the Xho I and Not I restriction sites (optimized MiAMP1 gene, described in SEQ ID NO: 1) are double digested, the double digested product is coagulated with 1% agarose Gel electrophoresis detection and use of DNA gel recovery kit to recover the optimized target fragment and expression vector. The recovered target fragment was ligated by T4 ligase at 4°C for 16 hours.

[0049] The double digestion system is:

[0050]

[0051] After recovering the target fragment and linearized expression vector, the reaction system for overnight ligation is:

[0052]

[0053] (2) The overnight reaction system...

Embodiment 2

[0063] Example 2. Transformation and identification of recombinant GS115 / MiAMP1 yeast

[0064] 1. Experimental materials:

[0065] Recombinant expression vector pPICZαA / MiAMP1

[0066] Plasmid pPICZαA

[0067] Pichia pastoris GS115

[0068] Restriction endonuclease Sac I;

[0069] 2. Treatment:

[0070] (1) The recombinant expression vector pPICZα A / MiAMP1 and the empty vector pPICZα A without the introduction of antimicrobial peptides were digested with Sac I.

[0071] (2) Take 10μL of the linearized vector and 80μL of the prepared GS115 competent cells, mix them and transfer them to the electroporation cup. According to the procedures set by the Bio-Rad electroporator, they will be transformed into competent yeast cells by electroshock, and 1mL of 1M is quickly added. Sorbitol was allowed to stand at 28°C for 1 hour, and 200μl of electroporation conversion solution was evenly spread on YPD+Zeocin resistant plates (Zeocin concentration 100μg / mL), and cultured at 28°C until a single colon...

Embodiment 3

[0078] Example 3. Expression and identification of MiAMP1 in yeast

[0079] 1. The expression level of MiAMP1 in recombinant yeast GS115 / MiAMP1

[0080] 1. Experimental materials:

[0081] Recombinant yeast GS115 / MiAMP1

[0082] Bradford protein concentration determination kit;

[0083] 2. Treatment:

[0084] (1) Pick a single colony of the recombinant strain GS115 / MiAMP1 that has been verified that the target gene has been integrated into the yeast genome and the empty plasmid control strain GS115 / pPICZαA to induce expression. Methanol was added every 24 hours to a final concentration of 1% for continuous induction for 108 hours; 1 mL samples were taken every 12 hours and stored for determination of antimicrobial peptide expression levels.

[0085] (2) Centrifuge the sample at 8000g for 10 minutes, collect the supernatant and the precipitate, and take 50uL of the supernatant at each sampling time point to detect the protein concentration in the sample by the Braford method.

[0086] 3. R...

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Abstract

The invention discloses a preparation GS115 / MiAMP1 for inhibiting pear-fruit postharvest penicilliosis. The preparation is recombinant yeast GS115 / MiAMP1 obtained after MiAMP1 antimicrobial peptide isintroduced. A construction method for the recombinant yeast GS115 / MiAMP1 includes the following steps that optimized MiAMP1 genes are constructed onto pPICZalphaA vectors through Xho I and Not I double enzyme digestion and T4 ligase, the formed pPICZalphaA / MiAMP1 recombinant expression vectors are subjected to linear enzyme digestion, and then are electrically transformed into GS115 competent cells, a single colony PCR is selected in a Zeocin-contained resistant plate, sequencing verification is carried out, and a recombinant pichia yeast GS115 / MiAMP1 strain is obtained. The preparation GS115 / MiAMP1 can be used for refreshment for inhibiting fruit postharvest diseases.

Description

Technical field [0001] The invention relates to the technical field of prevention and control of fruit diseases after harvest, and more specifically is a technique for transforming and overexpressing MiAMP1 antibacterial peptides in Pichia pastoris GS115 to improve the prevention and treatment of fruit diseases. Background technique [0002] At this stage, chemical fungicides are mainly used to prevent and control the diseases of fruits and vegetables. However, excessive use of chemical fungicides can endanger human health, cause environmental pollution, and develop drug resistance. At present, the European Union has banned the application of chemical fungicides in stone fruit. Therefore, it is an urgent need to find safe, non-toxic, environmentally friendly and highly effective antibacterial chemical fungicides. [0003] The use of antagonistic microorganisms for the biological control of postharvest fruit diseases is regarded as one of the most likely alternatives to chemical fun...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/29C12N15/81A23B7/155C12R1/84
CPCA23B7/155C07K14/415C12N15/815C12N2800/22
Inventor 余挺林明黄伊宁郑晓冬
Owner ZHEJIANG UNIV
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