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Method for obtaining positive transformant of thermomyces fungi

A technology of transformants and fungi, applied in the field of obtaining positive transformants of thermophilic fungi, can solve the problems of target compound discovery, in-depth study of biosynthetic pathways, rational design and directional synthesis difficulties, etc., to achieve scale and high-throughput The effect of quantitative screening

Active Publication Date: 2018-10-02
YUNNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the genetic transformation of thermophilic fungi has always been a technical problem in this field, there has been no report on the application of protoplast transformation to the study of biosynthetic genes of thermophilic fungi.
Limited by genetic manipulation, culture conditions, detection methods, etc., it is difficult to discover target compounds, in-depth study of biosynthetic pathways, rational design and directional synthesis

Method used

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  • Method for obtaining positive transformant of thermomyces fungi
  • Method for obtaining positive transformant of thermomyces fungi
  • Method for obtaining positive transformant of thermomyces fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Construction of knockout plasmids

[0067]The full-length knockout fragment (SEQ ID No.1) was constructed by constructing homologous recombination to inactivate the key functional domain KS in the gene Talth1_006666_t1 encoding PKS in the genome of Dupont thermophila NRRL 2155.

[0068] Using the thermophilic Dupont bacteria NRRL 2155 genome as a template, the upstream homology arm fragment and the downstream homology arm fragment of the KS gene in Talth1_006666_t1 were obtained by PCR method, wherein KS31up-F (SEQ ID No.2, 5' end contains SbfI restriction site, the restriction site and its upstream (5' end direction) 25bp and the pPK2.SUR.eGFP vector after digestion with SbfI (given to Liang Lianming, State Key Laboratory of Biological Resources Utilization and Conservation, Yunnan Associate researcher) sequence overlapping at one end) and KS31up-R (SEQ ID No.3, 5' end contains a 20bp DNA sequence overlapping with the hygromycin resistance gene expression cassette frag...

Embodiment 2

[0070] By constructing the full-length homologous recombination knockout fragment (SEQ ID No.10), the key functional domain KS in the gene Talth1_002859_t1 encoding PKS in the genome of Dupont thermophila NRRL 2155 was inactivated.

[0071] Using the Dupont thermophilic NRRL 2155 genome as a template, the upstream and downstream homology arm fragments of the KS gene in Talth1_002859_t1 were obtained by PCR, wherein, KS12up-F (SEQ ID No.11, 5' end contains the Sbf I restriction site point, the restriction site and its upstream (5' end direction) 25bp overlap with the 25bp sequence at one end of the pPK2.SUR.eGFP vector after digestion with Sbf I) and KS12up-R (SEQ ID No.12, 5' The 25bp DNA sequence overlapping with the hygromycin gene expression cassette at the end) was used as a primer pair to carry out PCR to obtain the upstream homology arm fragment of the KS gene in Talth1_002859_t1, and the KS12down-F (SEQ ID No.13, 5' end containing and anti-hygromycin Hygromycin gene exp...

Embodiment 3

[0073] Preparation of protoplasts from Dupontella thermophile NRRL 2155

[0074] (1) Inoculate the mycelium block of Dupont thermophilic bacteria NRRL 2155 in the center of the PDA medium, and place it in an incubator at 45°C for 7 days. Use 1mL pipette tip and sterile water (add 0.05% Tween 20) to scrape the culture from the above plate, filter with four layers of lens paper, and divide the liquid containing spores into 1.5mL centrifuge tubes, 10000rpm , enrich the spores by centrifugation at room temperature for 5 minutes, discard the supernatant, and enrich to 10 8 cells / mL and washed twice with sterile water.

[0075] (2) Transfer 200 μL of the spore suspension to 100 mL of YPS liquid medium, culture at 45° C. and shake at 180 rpm for 20 h.

[0076] (3) Pour the mycelium cultivated in step (2) into a sterile funnel (containing four layers of lens-cleaning paper) and filter to collect the mycelium.

[0077] (4) Wash the mycelia twice with P buffer solution.

[0078] (5)...

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Abstract

The invention provides a method for obtaining a positive transformant of thermomyces fungi. The method comprises the following steps: 1) preparing a protoplast solution of the thermomyces fungi; 2) mixing the protoplast solution and a homologous recombinant fragment to obtain a first mixture; 3) performing primary ice bath on the first mixture, then adding a PTC conversion solution, culturing andthen performing secondary ice bath to obtain a second mixture; 4) coating a solid culture medium with the second mixture for culture to obtain the at least one to-be-identified transformant; and 5) performing subculture on the at least one to-be-identified transformant to obtain a subculture product and performing DNA level identification on the subculture product so as to screen out the positivetransformant of the thermomyces fungi.

Description

technical field [0001] The present application provides a method for obtaining positive transformants of thermophilic fungi. Background technique [0002] Thermomyces fungi are a group of fungi that are suitable for growth at a temperature of 40°C to 50°C, with a minimum growth temperature of 20°C and a maximum growth temperature of 60°C. At present, researches on thermophilic fungi at home and abroad focus on the thermostable xylanase, thermostable chitinase and lipase produced by them. As an important resource for the production of new active compounds, fungi grow in extreme environments. Fungi in , which represent secondary metabolites in thermophilic fungi, have been rarely studied. In 2012, Guo Jipeng et al. isolated six new skeleton PKS-NRPS hybrid biogenic thirteen-membered macrolide compounds Thermolides A-F from Thermomyces dupontii NRRL 2155, and two of them showed extremely Strong poisonous and nematode killing effect is equivalent to that of abamectin, a nemati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/90C12R1/645
CPCC12N15/902
Inventor 牛雪梅何佳宁张克勤
Owner YUNNAN UNIV
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