Construction and Application of Heterologous Expression System of Aspergillus nidulans
A heterologous expression technology of Aspergillus nidulans, applied in the biological field, can solve the problems of slow growth, unclear genetic background and great difficulty of Dendrosporium, and achieve the effect of high-efficiency expression production
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Embodiment 1
[0037] Embodiment 1 Construction of Aspergillus nidulans proteolytic enzyme deletion strain
[0038] Aspergillus nidulans was used as the starting strain for genetic modification. This example uses high-fidelity enzyme PCR to amplify the pyroA auxotrophic marker gene sequence and the Aspergillus nidulans proteolytic enzyme gene dppV, mnn9 and pepA upstream and downstream 1300bp sequences, and then uses the Double-joint PCR method to construct the Aspergillus nidulans proteolytic enzyme respectively Single-gene knockout cassettes for genes dppV, mnn9, and pepA, and gene knockout in Aspergillus nidulans using PEG-mediated protoplast genetic transformation.
[0039] The PEG-mediated protoplast genetic transformation method of Aspergillus nidulans is as follows:
[0040] The used Aspergillus nidulans strain activation culture medium of the present invention is GMM culture medium, and the described solid culture medium of every 1L is prepared as follows: glucose 10g, nitrate mothe...
Embodiment 2
[0052] The construction of embodiment 2 Aspergillus nidulans RsmA gene overexpression strain
[0053] Aspergillus nidulans was used as the starting strain for genetic modification. In this embodiment, first, high-fidelity enzyme PCR is used to amplify the pyrG auxotrophic marker gene, the constitutive strong promoter gpdA promoter sequence, the upstream 1300bp sequence of the RsmA gene and the 1300bp gene fragment of the RsmA gene, and then the double-joint PCR method is used to amplify pyrG auxotrophic marker gene and constitutive strong promoter gpdA promoter construct the overexpression cassette of Aspergillus nidulans RsmA gene, and utilize the protoplast genetic transformation method mediated by PEG to carry out inheritance transformation to Aspergillus nidulans (transformation method is the same as embodiment 1), complete homologous recombination and carry out the overexpression transformation of the RsmA gene in the bacterium, then pick the transformant and extract geno...
Embodiment 3
[0054] Example 3 Construction of laccase-related expression vectors and aurovertin biosynthesis-related expression vectors
[0055] The laccase gene used in this application (as shown in SEQ ID NO: 1) comes from Pycnoporus sanguineus (GenBank: AY510604.1), using Pycnoporus sanguineus genomic DNA as a template, and using high-fidelity enzymes for PCR amplification , respectively cloned into the expression cassette (3476bp) and the laccase gene containing its own promoter sequence of the laccase gene, its terminator sequence, and at the same time using Aspergillus nidulans genomic DNA as a template, using high-fidelity enzymes for PCR amplification, respectively cloned to the gpdA promoter (1513bp) and aflR promoter (660bp) sequences; then use the conventional enzyme-cut ligation method and the Quick-change vector construction technology method to transform Escherichia coli and construct a vector containing the AMA1 autonomous replication sequence (see literature: Aleksenko A, C...
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