Botrytis cinerea mutant strain [delta]bcscd1 capable of producing scytalone and construction method thereof

A technology of cylindroxone and its construction method, which is applied in the biological field and can solve the problems of poor extraction effect, cumbersome operation, and high time and cost of mycelia processing

Inactive Publication Date: 2017-12-26
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The disadvantages of these methods include: First, the time and cost of mycelium treatment is high. Culture in a constant temperature shaker at 28°C and 120r / min for 7 days, then filter the mycelium, and dry in an oven at 70-75°C for 48h
Second, the extraction is time-consuming and cumbersome. During the extraction, the extraction is carried out in an autoclave at 121°C for 20 minutes, in a boiling water bath for 2 hours, and in a boiling water bath for 5 hours. During the boiling water bath, it is necessary to pay attention to supplementing NaOH, and shake the vial every 30 minutes or so
Third, the extraction effect is poor, and the extraction effect of mycelia melanin is 5h in boiling water bath > 2h in boiling water bath > 20min at 121°C
Among the three high-temperature extraction conditions, the extraction in a high-temperature sterilization pot at 121°C for 20 minutes is the most convenient, but the extraction effect is poor and the dissolved melanin is less
[0008] No method has been reported to obtain a large amount of fungal melanin or related intermediates

Method used

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  • Botrytis cinerea mutant strain [delta]bcscd1 capable of producing scytalone and construction method thereof
  • Botrytis cinerea mutant strain [delta]bcscd1 capable of producing scytalone and construction method thereof
  • Botrytis cinerea mutant strain [delta]bcscd1 capable of producing scytalone and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0114] Example 1, the sequence cloning of the bcscd1 encoding gene bcscd1 of Botrytis cinerea cinerea

[0115] The wild-type bacterial strain B05.10 used in the present invention is a reference strain of wild-type Botrytis cinerea. Firstly, find the gene sequence of the cylindroxone dehydratase coding gene Bcscd1 of Botrytis cinerea standard strain B05.10 from the Botrytis cinerea genome database (Ensembl Fungi), and design primers in the non-coding region of the gene expression frame, and use wild-type The DNA of B05.10 was used as a template, and high-fidelity enzymes were used for PCR amplification, and the amplified products were sequenced by the company.

Embodiment 2

[0116] Example 2, construction of knockout fragments

[0117] By means of homologous recombination, the gene Bcscd1 encoding cylindroxone dehydratase was replaced and knocked out with the hygromycin resistance gene Hph. The specific implementation steps are as follows:

[0118] Using the wild-type B05.10 DNA as a template, design specific primers Bcscd1-U-F and Bcscd1-U-R (Table 1) in the upstream non-coding region of the gene bcscd1, and use this pair of primers and PCR technology to amplify the Bcscd1 initiation codon The front non-coding region 5'Bcscd1 of 1007bp; with wild-type B05.10 DNA as a template, design specific primers Bcscd1-D-F and Bcscd1-D-R (Table 1) in the downstream non-coding region of gene Bcscd1, using this pair Primers and PCR techniques amplify the non-coding region (1122 bp) downstream of the Bcscd1 stop codon 3'Bcscd1; using the plasmid P22 as a template, use primers Hph-F and Hph-R (Table 1) to amplify the 5' and 3' two The hygromycin resistance gen...

Embodiment 3

[0120] Embodiment 3, acquisition and identification of mutant strains

[0121] Transfer the single colony (transformant) grown on the transformation upper layer plate to a PDA plate containing 75mg / l hygromycin, culture at 23±1°C, pick the youngest mycelia at the edge of the colony the next day, and transfer to a new On a PDA plate containing 75 mg / l hygromycin, subculture for 3-4 generations until the phenotype of the transformant is stable. At the DNA level, use the target gene internal primer, hygromycin internal primer, target gene upstream homology arm external primer / hygromycin internal primer and hygromycin internal primer / target gene downstream homologous arm external primer to identify mutant strains, Genomic DNA was extracted by the CTAB method, and the transformants verified at the DNA level without the target gene but with the correct insertion of the transformed fragment were selected. For the identification results, see image 3 , the characteristic bands of hyg...

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Abstract

The invention belongs to the field of biological technology. The wild-type cinerea cinerea strain is used as the background strain to knock out the gene of a key enzyme cylindroxone dehydratase in the melanin synthesis pathway, and construct a gene deletion mutation of the cinerea cinerea capable of producing a large amount of cylindroxone The main steps of the strain Δbcscd1 are as follows: use fusion PCR technology to fuse the 5'-UTR and 3'-UTR of the target gene bcscd1 with the upstream and downstream coding regions of the hygromycin fragment Hph to obtain the upstream and downstream fusion fragments, and use PEG-mediated The protoplast transformation method was used to transform the fusion fragment into wild-type protoplasts for double-crossover homologous recombination, and the gene deletion mutant strain Δbcscd1 was obtained through screening and identification. The gene deletion mutant strain constructed by the method of the invention has high genetic stability and low toxicity. The present invention also proposes a method for specifically purifying cylindroporin through steps such as extraction, rotary steaming and column chromatography.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of a Botrytis cinerea gene deletion mutant strain Δbcscd1 capable of producing scytalone in large quantities, and a method for separating and purifying scytalone. Background technique [0002] Melanin is a polymer formed by oxidative polymerization of phenolic or indole substances, which can be produced by bacteria, fungi, plants and animals. Studies have shown that the natural physical and chemical properties of melanin endow melanin with many common biological functions, such as anti-ultraviolet radiation, anti-oxidation, anti-hydrolytic enzymes, binding heavy metals, anti-fungal agents, anti-extreme temperature and drought, etc. These functions make melanin play an important role in the survival and self-protection of organisms. Therefore, the melanin synthesized by fungi plays an important role in their physiological activities. [0003] Fungal mela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12P7/26C07C45/78C07C45/79C07C49/747G01N30/90C12R1/645
CPCC12N9/88C07C45/78C07C45/79C12P7/26C12Y402/01094G01N30/90
Inventor 朱品宽向升何一凡张成花许玲
Owner EAST CHINA NORMAL UNIV
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