Alpha-glucosidase gene knockout Aspergillus niger strain and application thereof
A technology of glucosidase and Aspergillus niger strain, applied in the field of genetic engineering, can solve the problems of affecting the activity of glucoamylase and low efficiency
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Embodiment 1
[0046] [Example 1] Amplification of Aspergillus niger α-glucosidase gene α-gluE1, α-gluE2 and selectable marker gene
[0047] Genomic DNA of Aspergillus niger HE01 was extracted with an OMEGA kit, and used as a template, with GLU-1F and GLU-2R as a pair of primers upstream of the α-glucosidase gene (GenBank: D45356.1), and with GLU-3F, GLU-2R GLU-4R is a pair of primers downstream of the α-glucosidase gene, using Trans The FastPfu Fly DNA Polymerase PCR system amplified the α-gluE1 (SEQ ID NO.19) about 1 000 bp upstream of the α-glucosidase gene and the α-gluE2 (SEQ ID NO.19) about 1 000 bp downstream of the α-glucosidase gene. 20). The target fragment obtained by PCR was subjected to 1% agarose gel electrophoresis, and the fragment size was the same as expected. After comparing the sequence with DNAsist software, it was found that it was identical to the upstream and downstream of the Aspergillus niger α-glucosidase gene recorded in (GenBank D45356.1). The homology of the 1...
Embodiment 2
[0056] [Example 2] Construction of Knockout Expression Cassette
[0057] The expression cassette to be constructed in this example is α-gluE1-N R (SEQ ID NO.22) and N R -α-gluE2 (SEQ ID NO.23), the above-mentioned PCR amplifies to three gene fragments α-gluE1, α-gluE2, N R Afterwards, they were purified and recovered separately, and α-gluE1-N was obtained by Overlap PCR R and N R -Two fusion fragments of α-gluE2, as a linear expression cassette for gene knockout, the specific steps are as follows:
[0058] With α-gluE1 and N R The two fragments are used as templates, and GLU-1F and GLU_MkR are used as upstream and downstream primers to amplify α-gluE1-N R Fusion fragment; α-gluE2 and N R The two fragments are templates, GLU_MkF and GLU-4R are upstream and downstream primers, and Go DNAPolymerase PCR system to amplify N R - α-gluE2 fusion fragment.
[0059] Overlap PCR system:
[0060]
[0061]
[0062] The two fusion fragments α-gluE1-N obtained by the above P...
Embodiment 3
[0063] [Example 3] Protoplast transformation of Aspergillus niger HE01 and identification of recombinant Aspergillus niger HE01
[0064] 1. The linear expression cassette was co-transformed into Aspergillus niger HE01
[0065] Extraction of α-gluE1-N from Escherichia coli R and N R -The two fusion fragments of α-gluE2 are respectively connected to the circular cloning vector of pMD19-T-Vector (simple), and a large amount of α-gluE1-N can be obtained by PCR amplification R and N R - α-gluE2 fragment, linearized expression cassette is beneficial to improve protoplast transformation rate. After the protoplasts were transformed into Aspergillus niger HE01 and regenerated, the overnight-grown protoplast bacteria liquid was evenly spread on 5 to 6 large plates (8.5 cm) of nourthricin sulfate with a concentration of 125 μg / mL, and 32 Cultivate at ℃. The linear expression cassette undergoes homologous recombination with the strain genome in the nucleus, so that the positive strai...
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