三种白腐菌生物学特性与木质纤维素酶基因遗传多样性

以采自东北林业大学帽儿山实验林场的3种白腐真菌——木蹄层孔菌(Fomes fomentarius)、鲍姆鲍姆木层孔菌(Phellinus baumiibaumii)和火木层孔菌(Phellinus igniarius)为材料,用菌落直径测量法比较3种白腐菌在马铃署葡萄糖固体培养基上的生长速度,采用菌丝体干重法比较其在马铃署葡萄糖液体培养基中的生物量变化。结果显示:马铃薯葡萄糖固体培养基上3种白腐菌均为快速生长类型,其生长速度木蹄层孔菌火木层孔菌鲍姆鲍姆木层孔菌;马铃署葡萄糖液体培养基中生物量增长速度木蹄层孔菌鲍姆鲍姆木层孔菌火木层孔菌。用比色法测量其木质纤维素酶活性,结果显示:木蹄层孔菌产锰过氧化物酶和漆酶量较高,鲍姆鲍姆木层孔菌和火木层孔菌产木质素过氧化物酶量较高;木蹄层孔菌、鲍姆鲍姆木层孔菌和火木层孔菌3种白腐菌的3种主要木质素酶(锰过氧化物酶、漆酶和木质素过氧化物酶)的表达量,种间差异显著(F=3.75*、5.20**、3.01*),白桦木屑诱导处理与对照间差异显著(F=3.84*、4.19*、5.28*);两种主要纤维素酶(葡聚糖内切酶、葡聚糖外切酶)的表达量,种间差异不显著,受碳源影响作用显著(F=3.99*、4.04*)。筛选29对引物组合,对3种白腐菌几种主要木质纤维素酶基因进行TRAP-PCR分子标记检测,比较3菌种间遗传差异,扩增总条带数为357条,多态性条带数为255条,多态性条带的比例为71.43%,其中木质素降解酶基因总多态位点比率为73.77%,纤维素降解酶基因总多态位点比率为68.97%。3种白腐菌的木质纤维素降解酶基因在种间均存在较高的遗传差异。因此,特定基因的TRAP分子标记可以用于木腐菌的遗传变异分析。

Biological characteristics of three white-rot fungi and genetic diversity of lignocellulose enzyme related genes

We used three types of white-rot fungi, namely, Fomes fomentarius, Phellinus baumiibaumii, and Phellinus igniarius, as the study materials. The colony diameters and mycelial dry weights in different culture media were measured to compare their growth characteristics. Further, the activities of five lignocellulose degradation enzymes of the fungi were detected using colorimetry. The results showed that all the fungi were fast growing types:colony growth of P. baumii baumii in Potato Dextrose Agar medium was faster than that of P. igniarius and F. fomentarius, but mycelial biomass accumulation of F. fomentarius in liquid Potato Dextrose medium was the highest, followed by P. baumii baumii and P. igniarius. The manganese peroxidase (MnP) and laccase (Lac) productions of F. fomentarius were higher than that of the other two species, while the lignin peroxidase (Lip) yields of P. baumii baumii and P. igniarius were higher than that of F. fomentarius. The F-test value of three ligninolytic enzymes expressions among the three fungi species was 3.75^*, 5.20^{* *} and 3.01^* for lignin peroxidase, manganese peroxidase and laccase respectively, and the value for manganese peroxidase reaching a highly significant level. The wood chip induced treatment had significant difference from the control, and F-test value for lignin peroxidase, manganese peroxidase and laccase was 3.84^*, 4.19^* and 5.28^* respectively. No clear variations among fungi species for the expressions of endoglucanase and exoglucanase were noted, while the expressions were obviously affected by the carbon source in the medium, and the F-test value was 3.99^* and 4.04^* for endoglucanase and exoglucanase respectively, both values reached a significant level. Target region amplification polymorphism (TRAP) was used to analyze the lignocellulose degradation enzyme gene polymorphisms in the three white-rot fungi by using 29 sets of selected primers. By using TRAP, we amplified 357 bands, including 255 polymorphic bands, with 71.43% of the total polymorphic loci percentage, where the polymorphic loci percentage of the lignin and cellulose degradation enzyme genes reached 73.77% and 68.97%, respectively. This result indicated that the lignocellulose degradation enzyme genes of the three white-rot fungi have high interspecific variations. Thus, we showed that TRAP molecular markers are useful for the analysis of variations among species of wood-rot fungi at the gene level.