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

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...

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

Images