层出镰孢菌原生质体制备条件的研究

Study on Preparation Conditions of Protoplast of Fusarium proliferatum

[目的]研究层出镰孢菌(Fusarium proliferatum)原生质体的最佳条件,建立高效制备层出镰孢菌原生质体制备的方法.[方法]通过对菌丝体菌龄、酶解液组合、酶解反应温度及酶解时间等影响因素进行优化,对层出镰孢菌原生质体的制备条件进行研究.[结果]菌块在CMC液体培养基中25℃培养分生孢子3 d后,过滤离心后转移至YEPD液体培养基中培养12 h,该菌丝最适于层出镰孢菌原生质体的制备;用1.2 mol/L KCl溶液配制含有5 mg/m L裂解酶、25 mg/m L崩溃酶、0.05 mg/m L溶壁酶的酶解液在30℃下对菌丝酶解1.5 h,此时原生质体的数量最大,为2.47×10~9个/m L.原生质体再生培养时,使用40%PTC溶液恢复细胞壁,在TB3液体培养基中30℃过夜培养,离心后与TB3固体培养基混合后倒入平板培养,原生质体再生率可达39.75%.[结论]通过对菌丝体菌龄、酶解液浓度、温度、酶解时间等条件的优化,可提高层出镰孢菌原生质体的产量及再生率,为进一步研究层出镰孢菌致病分子机制提供理论依据.

[ Objective] The aim was to study the optimum conditions for the preparation of protoplasts of Fusarium proliferatum, and to estab-lish efficient methods for preparing protoplast. [ Method ] The preparation conditions of protoplast of Fusarium proliferatum were studied by op-timizing the factors including the hypha culture time, enzyme mixtures, enzymolysis reaction temperature and enzymolysis time. [Result] The result showed that mycelia was cultured in CMC liquid medium for three days at 25 °C , and then the conidia was transferred to YEPD liquid medium for 12 hours, which was suitable for the preparation of protoplast of Fusarium p r o l i f e r a t u m enzyme mixture containing 5 mg/mL lysing enzyme, 25 mg/mL driselase and 0.05 mg/mL lywallzyme was dissolved in 1.2 moi/L KC1 solution;The enzymolysis reaction tempera-ture was 30 °C and enzymolysis time was 1. 5 h, the yield of protoplast was the highest, and the number of protoplasts was 2.47 x l〇 9 proto-plasts/mL. When the protoplast was regenerated, 40% PTC solution was used to restore the cell wall, and incubated in TB3 liquid medium at 30 °C overnight. After centrifugation, the cells were mixed with TB3 solid medium and poured into plate culture. The protoplast regeneration rate was 39. 75% . [ Conclusion ] The yield and regeneration rate of protoplast of Fusarium proliferatum were improved by optimizing the condi-tions such as hypha culture time, enzyme mixtures, enzymolysis reaction temperature and enzymolysis time, and also provide theory basis for further study on pathogenicity molecular mechanism of Fusarium proliferatum.