| Popis: |
In order to obtain the optimal conditions for the protoplast preparation of Pleurotus giganteus, two strains, PG46 and PG79, with different temperature types, were selected as materials to study the effects of five factors (mycelial age, osmotic stabilizer type, lywallzyme concentration, enzymatic hydrolysis duration and enzymatic hydrolysis temperature) based on single factor and orthogonal experimental methods. The results were as follows: (1) In the single factor test, the optimal conditions for the protoplast preparation of P. giganteus were mycelial culture for 5 d, using 2.5% lywallzyme with 0.6 mol·L-1 mannitol, incubated for 4 h at 32℃ (PG46) or 27-35℃ (PG79). (2) Orthogonal experiment verified and optimized the single factor test results. Combination 2 (mycelial age 5 d, lywallzyme concentration 2.5%, 0.6 mol·L-1 mannitol, incubated for 4 h at 32℃) could be used as the optimal condition for the protoplast preparation of PG46 and PG79 at the same time, and the protoplast yields were 11.22 × 106 CFU·mL-1 and 7.28 × 106 CFU·mL-1, respectively. (3) For F-test, the influence degree of various factor on the protoplast preparation were mycelial age>lywallzyme concentration>enzymatic hydrolysis temperature>enzymatic hydrolysis duration (PG46), and mycelial age>enzymatic hydrolysis duration>enzymatic hydrolysis temperature>lywallzyme concentration (PG79), respectively. In conclusion, the protoplast preparation conditions of the two P. giganteus strains with different temperature types were basically the same, and the effects of mycelial age on the protoplasts yield of the two strains were the most significant. The results can lay a foundation for further cross-breeding, genetic transformation, whole genome sequencing and promote the molecular genetics development of P. giganteus. |
