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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 number of protoplasts and mycelial cell wall degradation of enzymatic hydrolysis of the predatory fungi Duddingtonia flagrans by using enzymatic hydrolysis method, the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) The protoplasts produced by enzymatic digestion of the predominant fungi were fluorescently labeled. The effect of labeling concentration, labeling time and incubation temperature on protoplast labeling were investigated respectively. The regeneration of protoplasts after CFSE labeling was also observed. The results showed that the final concentration of CFSE was 10μmol / L, the labeling time was 15min and the incubation temperature was 36 ℃, which was the ideal condition for the CFSE labeling of protoplasts. Simultaneously, CFSE did not affect the regeneration rate of protoplasts. CFSE, as a fluorescent probe of living cell tracer, can rapidly and efficiently label the protoplasts of predatory fungi. This method provides a new idea for the rapid evaluation of the preparation quality of the predominant fungal protoplasts.