Preparation and regeneration method of muscodor protoplast
A technology of protoplasts and musk mold, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve the problems of slow growth of strains, achieve the effect of shortening the time required and improving the transformation efficiency
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Embodiment 1
[0052] A method for preparing and regenerating musk mold protoplasts, comprising the steps of:
[0053] Step 1, activate the bacteria:
[0054] Thaw the cryopreservation tube containing musk mold ZJLQ024 naturally at room temperature, use a sterilized toothpick to take out the bacteria block from the tube, inoculate it into PDA medium, seal it with a parafilm, and culture it in the dark in a 25°C incubator;
[0055] Wherein the musk mold strain ZJLQ024 bacterial classification is derived from Zhejiang University College of Agriculture and Biotechnology, and is preserved in China General Microorganism Culture Collection and Management Center (preservation number: CGMCC2863).
[0056] PDA medium: 200g potato, 20g glucose, 18% agar powder, 1000mL pure water, autoclaved at 121℃ for 15min;
[0057] Step 2, prepare seed culture solution:
[0058]In the ultra-clean workbench, wash the hyphae on the surface of the ZJLQ024 colony cultivated in the incubator at 25°C for a suitable tim...
Embodiment 2
[0072] The shaking culture time in Step 2 of Example 1 was replaced by 4d, 5d, 6d and 7d respectively, and the production of protoplasts was observed.
[0073] Analysis of the results: There were significant differences in the number of protoplasts prepared at different bacterial ages. The number of protoplasts prepared from the mycelium liquid cultured on a shaker for 4 days was 2.8×10 6 individual / mL. The number of protoplasts prepared from the mycelial liquid cultured for 5d and 6d increased compared with 4d, respectively 6.5×10 6 pcs / mL, 10.3×10 6 individual / mL. The number of protoplasts prepared when the bacterial age was 7 days was significantly reduced compared with the bacterial age of 6 days ( figure 2 ).
Embodiment 3
[0075] The lyase in Step 3 of Example 1 was replaced by lysozyme, collapse enzyme, β-glucanase and compound enzyme respectively, and the production of protoplasts was observed.
[0076] Result analysis: when using a single lytic enzyme, the number of protoplasts prepared was significantly more than that prepared by using other enzyme solutions, reaching 10.5×10 6 individual / mL. The number of protoplasts prepared by using a single β-glucanase and collapsing enzyme was small, and the number of protoplasts prepared by mixing the above two enzymes with lysozyme increased compared with the use of a single enzyme. The number of protoplasts prepared by the mixed enzyme solution of glycanase and lysozyme reached 6.6×10 6 individual / mL; the number of protoplasts prepared by the mixed enzyme solution of collapsing enzyme and lysozyme reached 8.2×10 6 individual / mL. The number of protoplasts prepared by the mixed enzyme solution of the three enzymes was 8.1×10 6 pcs / mL( image 3 -a)...
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