Functional protein TP06128 and coding gene and application thereof
A gene and encoding technology, applied in the field of functional protein TP06128 and its encoding gene and application, can solve the problems of low yield and high cost of biomass degrading enzymes
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Embodiment 1
[0121] Example 1, Discovery of the functional protein TP06128 and its coding gene of Pinophilus pinophilum
[0122] Through extensive analysis and functional verification of the genome of Talaromyces pinophilus 1-95 and its transcriptome under different carbon source culture conditions, a new protein TP06128 and its coding gene were discovered.
[0123] The protein shown in Sequence 1 of the Sequence Listing was named TP06128 and consisted of 861 amino acid residues. The gene encoding TP06128 is named TP06128 gene, and the open reading frame (ORF) of the TP06128 gene is shown in sequence 2 of the sequence listing. The genomic DNA of the TP06128 gene is shown in sequence 3 of the sequence listing.
Embodiment 2
[0124] Embodiment 2, the construction of pinophilus TP06128 gene knockout cassette
[0125] 1. Extract the genomic DNA of Pinophilus 1-95.
[0126] 2. Using the genomic DNA obtained in step 1 as a template, the primer pair consisting of primer TP06128_left-arm-F and primer TP06128_left-arm-R was used for PCR amplification to obtain a 1956bp fragment upstream of the ORF of the TP06128 gene (see figure 1 Lane 3), called TP06128 left arm.
[0127] TP06128_left-arm-F: 5'-TCGTGTCATTCTTCCAGGGTT-3';
[0128] TP06128_left-arm-R: 5'-GTCCCTGGAAAATCAACTACTCCATTCCAGAGGTCGAGTGAGTA-3'.
[0129] 3. Using the genomic DNA obtained in step 1 as a template, the primer pair consisting of primer TP06128_right-arm-F and primer TP06128_right-arm-R was used for PCR amplification to obtain a 2661bp fragment downstream of the ORF of the TP06128 gene (see figure 1 Lane 1), called TP06128 right arm.
[0130] TP06128_right-arm-F: 5'-GATCCCCCGGGGATCCACAACTATCTGTTTGAATATTTGAG-3';
[0131] TP06128_rig...
Embodiment 3
[0143] Example 3. Construction and verification of the gene deletion mutant strain ΔTP06128 of Pinophilus pinophilum TP06128
[0144] 1. Taking P. pinophilum 1-95 as the starting strain, knocking out its TpKu70 gene to obtain the mutant ΔTpKu70 of P. pinophilum.
[0145] The literature describing the specific steps of the pinophilus mutant ΔTpKu70 and knocking out the TpKu70 gene is as follows: Zhang T, et al. Deletion of TpKu70 facilitates gene targeting in Talaromycespinophilus and identification of TpAmyR involvement in amylase production[J].World Journal of Microbiology&Biotechnology , 2017, 33(9): 171. The name of the pinophilus mutant ΔTpKu70 in the literature is "Talaromycespinophilus mutant ΔTpKu70".
[0146] 2. Preparation of protoplasts of the pinophilus mutant strain ΔTpKu70
[0147] (1) Inoculate the pinophilus mutant ΔTpKu70 obtained in step 1 on a PDA medium plate, and after static culture at 28°C for 6 days, elute the spores on the surface of the plate with 0....
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