Thermoascus gene expression system
A thermophilic ascomycete and expression system technology, which is applied in the field of thermophilic ascomycete gene expression system and can solve the problem of no effective available genetic operating system and the like
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Embodiment 1
[0069] Example 1 Screening of pyrG gene-deficient strains
[0070] Wild-type Thermoascus NBRC 9748 was inoculated in MEA medium, cultured in a 37°C incubator for 7 days, and washed 3 times with 5 mL of normal saline to obtain a spore suspension. Count using a hemocytometer and adjust the concentration to 1 x 10 6 individual / mL. Then take 10mL and place it in a sterilized empty petri dish, place the petri dish at a distance of 30cm from a 20W ultraviolet lamp and irradiate it for 5 minutes. The spore suspension irradiated by ultraviolet was spread on the Chapei medium containing 0.3% 5'-fluoroorotic acid (5'-FOA) and 0.01% uracil for orotidine-5'-phosphate Decarboxylase gene (pyrG) deficient strains were screened to obtain uracil auxotrophic strains TA2 and TA3 ( figure 2 ), TA3 was selected for strain preservation and used for the construction of the subsequent expression system.
[0071] Cha's Medium
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Embodiment 2
[0073] Construction and Transformation of Embodiment 2 Vectors
[0074] (1) Amplification of pyrG gene and construction of expression vector
[0075] According to the pyrG amino acid conserved sequence DRKFIDI and PPTSSTAT of filamentous fungi, the primers Ta-pyrG-F2, Ta-pyrG-F3 and Ta-pyrG-R2 (see Table 1) were designed, and the genome of Thermoascomycetes was used as a template. The primers were used for PCR between different primer pairs, and a 400bp pyrG fragment was amplified and sequenced by primers pyrG-F3 and pyrG-R2. According to the obtained gene fragments, primers were designed for TAIL-PCR to obtain the DNA sequences of the 5' and 3' ends respectively, and a 1921 bp full-length sequence of the TA-pyrG gene was spliced.
[0076] The specific operation is:
[0077] (i) Cloning of pyrG gene fragment
[0078] PCR System Using Genomic DNA of Thermoascomycetes as Template
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[0080]
[0081] PCR program:
[0082]
[0083]The obtained PCR fragments we...
Embodiment 3
[0174] Induced expression of embodiment 3 amylase gene
[0175] CBHP-Aoamy-Tcbh-pyrG, CBHPS-Aoamy-Tcbh-pyrG, EGP-Aoamy-Tcbh-pyrG, EGPS-Aoamy-Tcbh-pyrG strains were inoculated in 100mL MEA medium (250mL Erlenmeyer flask), at 50°C , 200rpm shaking culture for 3 days, after 5000×g centrifugation mycelium was recovered and transferred into 100mL induction medium. At 50°C, 200rpm shaking induction culture for 4 days, the culture supernatant was collected for secreted expression (with signal peptide), and the mycelium for intracellular expression was recovered, resuspended in 8mL lysis buffer, and placed in a 130W sonicator Break for 10 minutes (break for 2 seconds, pause for 4 seconds), and then centrifuge at 12000g for 10 minutes in a centrifuge at 4°C. The supernatant was saved as an enzyme solution. Punch holes on the agar plate containing 1% soluble starch, then take 50 μl of the supernatant of extracellular secreted expression and 50 μl of broken solution of intracellular ex...
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