Recombinant Paecilomyces lilacinus strain pnvt8 and its application
A technology of Paecilomyces lilacinus and Paecilomyces lilacinus is applied in the field of agricultural microbial genetic engineering, which can solve different problems and achieve the effects of increasing growth rate, increasing sporulation rate, and increasing biomass and cell yield.
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Embodiment 1
[0046] Embodiment 1: recombinant Paecilomyces lilacinus strain
[0047] 1. Artificial design of vgb gene
[0048] Find the amino acid sequence of Vitreum hyaline hemoglobin (VHb) from GenBank (GenBank accession number: M27061), and then according to the codon degeneracy and the codon preference of Paecilomyces lilacinus (see URL
[0049] http: / / www.kazusa.or.jp / codon / codon usage database) principle, select the appropriate codon corresponding to each amino acid, and convert the amino acid sequence of VHb into the original nucleotide sequence. Then, use DNASIS software to analyze its repeat sequence and stem-loop structure, etc., through the replacement of local codons to make the length of the repeat sequence ≤ 7 bases, and the stem length in the stem-loop structure ≤ 7 bases, the final The nucleotide sequence of the vgb gene (such as figure 1 shown).
[0050] 2. Construction of fungal expression vector pUPNVT
[0051]The list of primers required for construction of the exp...
Embodiment 2
[0186] Embodiment 2: the fermentation performance detection of PNVT8
[0187] 1. The relationship between the expression of VHb protein and the amount of dissolved oxygen
[0188] In order to further determine the influence of dissolved oxygen conditions on the expression of VHb protein, the present invention determines the number of spores of ACSS and PNVT8 by a hemocytometer, and inserts 2.4×10 5 1 spore was cultured with gradient medium (20mL, 40mL, 60mL, 80mL, 100mL), sealed with a sealer, 200r / min, 28°C for 7 days, the experimental results are as follows Figure 14 shown.
[0189] Shake the above-cultured mycelia in turn, draw 5mL into a graduated plate, add 5mL of distilled water containing 1% Tween 80, mix well and count the mycelium balls that can be clearly seen by the naked eye. is the abscissa, and the number of hyphae balls is plotted as the ordinate. The experimental results are as follows Figure 15 shown
[0190] Depend on Figure 14 and Figure 15 It can ...
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