捕食性真菌Duddingtonia flagrans原生质体获得与再生的荧光标记评价

Fluorescence labeling of production and regeneration of protoplasts from the nematode-trapping fungus Duddingtonia flagrans

为了更准确快捷地评价利用酶解法酶解捕食性真菌Duddingtonia flagrans菌丝产生的原生质体数量及菌丝细胞壁降解情况,采用活体荧光染料羧基荧光素乙酰乙酸(carboxyfluorescein diacetate succinimidyl ester,CFSE),对该捕食性真菌菌丝酶解后产生的原生质体进行荧光标记,分别考察了标记浓度、标记时间、孵育温度对原生质体标记效果的影响,并观察CFSE标记后的原生质体再生情况。结果表明CFSE终浓度为10μmol/L,标记时间为15min,孵育温度为36℃,是CFSE标记捕食性真菌原生质体的理想条件,该试验同时表明CFSE不影响原生质体的再生率。CFSE作为一种活细胞示踪荧光探针,可以快速高效地标记捕食性真菌原生质体,该方法为捕食性真菌原生质体制备质量的快速评价提供了新的思路。

In order to evaluate the quantity of protoplasts and the degradation situation of the cell wall from the hyphae of nematode-trapping fungus Duddingtonia flagrans more accurately and quickly, the protoplasts of Duddingtonio flagrans produced by enzymolysis were labeled by a reactive fluorescent dye carboxyfluorescein diacetate succinimidyl ester （CFSE）. The optimal dosage and timing as well as the temperature of protoplast release and labeling were determined. Protoplast regeneration after labeling was also observed. The results indicate that the ideal conditions for protoplast labeling are as follows： the final dosage of CFSE is 10λmol/L, and labeling duration is 15min at the temperature of 36℃. In addition, the experiment showed that CFSE would not affect the regeneration rate of protoplast from Duddingtonia flagrans. The method provides an ideal technique for rapid evaluation of the protoplast-producing efficiency from the nematode-trapping fungus.