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Preparation gs115/ac-amp2 for inhibiting postharvest blue mold in pear fruit

A GS115, ac-amp2 technology, applied in the direction of virus/phage, microorganism-based method, application, etc., can solve the problem that the biological control effect of antagonistic microorganisms cannot be reached or approached, and achieves inhibition of penicillium. Occurrence and prolong shelf life , the effect of reducing costs

Inactive Publication Date: 2020-12-29
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/ac-amp2 for inhibiting postharvest blue mold in pear fruit
  • Preparation gs115/ac-amp2 for inhibiting postharvest blue mold in pear fruit
  • Preparation gs115/ac-amp2 for inhibiting postharvest blue mold in pear fruit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, recombinant expression vector construction

[0041] 1. Experimental materials:

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

[0043] Axygen DNA Gel Recovery Kit;

[0044] 2×Phanta Max Master Mix;

[0045] Escherichia coli competent cell DH5α;

[0046] Plasmid pPICZαA.

[0047] 2. Processing:

[0048] (1) After double digestion of the plasmid pPICZα A and the target gene (optimized Ac-AMP2 gene, described in SEQ ID NO: 1) containing Xho I and Not I restriction sites, the double digestion product was washed with 1% The optimized target fragment and expression vector were detected by agarose gel electrophoresis and recovered with a DNA gel recovery kit. The recovered target fragment was ligated by T4 ligase at 4°C for 16 hours.

[0049] The double enzyme digestion system is:

[0050]

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

[0052]

[0053...

Embodiment 2

[0064] Example 2. Transformation and Identification of Recombinant GS115 / Ac-AMP2 Yeast

[0065] 1. Experimental materials:

[0066] Recombinant expression vector pPICZα A / Ac-AMP2

[0067] Plasmid pPICZαA

[0068] Pichia pastoris GS115

[0069] Restriction enzyme Sac I

[0070] 2. Processing:

[0071] (1) The recombinant expression vector pPICZα A / Ac-AMP2, which was verified and successfully constructed above, and the empty vector pPICZα A without antimicrobial peptides were digested with Sac I.

[0072] (2) Mix 10 μL of the linearized carrier with 80 μL of the prepared GS115 competent cells and transfer them to the electroporation cup, transform them into competent yeast cells by electric shock according to the program set by the Bio-Rad electroporation instrument, and quickly add 1 mL of 1M Sorbitol, let stand at 28°C for 1 h, take 200 μl of electroporation transformation solution and spread evenly on YPD+Zeocin resistance plate (Zeocin concentration 100 μg / mL), and cult...

Embodiment 3

[0079] Example 3, Expression and Identification of Ac-AMP2 in Yeast

[0080] 1. Expression level of Ac-AMP2 in recombinant yeast GS115 / Ac-AMP2

[0081] 1. Experimental materials:

[0082] Recombinant Yeast GS115 / Ac-AMP2

[0083] Bradford protein concentration assay kit;

[0084] 2. Processing:

[0085] (1) Pick a single colony of the recombinant strain GS115 / Ac-AMP2 and the empty plasmid control strain GS115 / pPICZα A that has been verified that the target gene has been integrated into the yeast genome, and induce expression. Methanol was added every 24 hours to a final concentration of 1% and continued to induce for 108 hours; every 12 hours, 1 mL samples were taken and preserved to determine the expression level of antimicrobial peptides.

[0086] (2) The sample was centrifuged at 8000 g for 10 min, and the supernatant and precipitate were collected respectively. At each sampling time point, 50 uL of the supernatant was taken to detect the protein concentration in the sam...

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Abstract

The invention discloses a preparation GS115 / Ac-AMP2 for inhibiting pear-fruit postharvest penicilliosis. The preparation is recombinant yeast GS115 / Ac-AMP2 obtained after Ac-AMP2 antimicrobial peptideis introduced. A construction method for the recombinant yeast GS115 / Ac-AMP2 includes the following steps that optimized Ac-AMP2 genes are constructed onto pPICZalphaA vectors through Xho I and Not Idouble enzyme digestion and T4 ligase, the formed pPICZalphaA / Ac-AMP2 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 / Ac-AMP2 strain is obtained. The preparation GS115 / Ac-AMP2 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 postharvest fruit diseases, and more specifically relates to a technique for transforming and overexpressing Ac-AMP2 antibacterial peptides in Pichia pastoris GS115 to improve its prevention and control of fruit diseases. Background technique [0002] At present, chemical fungicides are mainly used to prevent and control post-disease diseases of fruits and vegetables. However, excessive use of chemical fungicides will endanger human health, cause environmental pollution, and produce drug resistance and other problems. Currently, the European Union has banned the application of chemical fungicides in drupe fruits, so it is an urgent need to find safe, non-toxic, environmentally friendly and highly effective chemical fungicide substitutes at this stage. [0003] The use of antagonistic microorganisms in the biological control of postharvest fruit diseases is considered to be one of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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