组织分离常用于食用菌菌种提纯和复壮,但连续多次组织分离会造成菌种退化。以草菇V844为初始菌株T0,连续组织分离继代18次获得继代菌株T1~T18,测定T0~T18的生理性状及纤维素、半纤维素、木质素降解相关酶活力,并结合转录组学分析。结果表明,随着继代次数的增加,草菇菌落直径逐渐变小,菌丝生长速度和菌丝生物质量先增加后减少。外切葡聚糖酶、漆酶、锰过氧化物酶和半纤维素酶活力先上升后下降,T18较T0分别下降了34.2%、71.1%、90.0%和58.7%;内切葡聚糖酶、滤纸酶和木聚糖酶活力在T0~T4差异不显著,之后开始下降,T18较T0分别下降了66.7%、86.4% 和67.9%;β-葡萄糖苷酶活力差异不显著(P>0.05)。对T0、T4、T8、T12和T16进行转录组学分析表明,与基质降解酶相关的差异表达基因有34个(上调8,下调17,先上调后下调9)。综合分析草菇组织分离继代菌株的生理性状、相关酶活力及转录组学发现,当连续组织分离继代4次时,草菇菌种有明显地复壮效果,但当连续继代12次以上时,组织分离继代会导致菌种出现退化现象。
Tissue separation is often used for purification and rejuvenation of edible fungi, but continuous tissue separation will lead to strain degradation. Using V844 as the initial strain T0, the subculture strain T1-T18 was obtained by continuous tissue isolation and subculture for 18 times. The physiological characteristics of T0-T18 and the enzyme activities related to cellulose, hemicellulose and lignin degradation were measured and analyzed by transcriptology. The results showed that with the increase of subculture time, the colony diameter of V. volvacea decreased gradually, and the mycelial growth rate and biological quality increased at first and then decreased. The activities of exoglucanase, laccase, manganese peroxidase and hemicellulase increased at first and then decreased. Compared with T0, those activities of T18 strain decreased by 34.2%, 71.1%, 90.0% and 58.7%, respectively. The activities of endoglucanase, filter paper enzyme and xylanase were not significantly different at T0-T4, and then began to decline. Compared with T0, T18 decreased by 66.7%, 86.4% and 67.9%, respectively. There was no significant difference in β-glucosidase activity (P>0.05). Transcriptomics analysis of T0, T4, T8, T12 and T16 showed that there were 34 differentially expressed genes related to matrix degradation enzymes (8 up-regulated,17 down-regulated, first up, then down 9). The physiological characters, related enzyme activities and transcription histology of the subculture of V. volvacea were analyzed synthetically. It was found that when the continuous tissue was separated and subcultured for four times, the strain of V. volvacea had obvious rejuvenation effect, but when the continuous subculture was more than 12 times, the tissue isolation and subculture would lead to the strain degradation.
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