双孢蘑菇对高温胁迫的响应及耐热机理

The response and thermotolerant mechanism of Agaricus bisporus under high temperature stress

本研究以双孢蘑菇Agaricus bisporus工厂化菌株A15和筛选得到的耐高温菌株A15-TH为研究对象,比较了高温胁迫对两个菌株菌丝生长的影响,并从氧化损伤修复及基础碳代谢-糖酵解途径两个角度探索双孢蘑菇对高温胁迫的响应及耐热机理。高温胁迫下,对照菌株A15的菌丝生长速度降低,菌丝分叉增加;而耐高温菌株A15-TH菌丝生长速度高于A15,菌丝形态优于对照菌株,表现出对高温具有一定的耐受性。对两个菌株高温胁迫下氧化损伤及抗氧化酶系统进行研究发现,高温胁迫30-90min导致对照菌株A15的三磷酸腺苷(ATP)含量下降54.4%-59.6%,线粒体复合物Ⅰ、Ⅱ、Ⅲ活性升高,超氧阴离子(O2-)含量增加了34.9%-71.3%;此外高温胁迫降低了超氧化物歧化酶(SOD)的活性,影响了O2-的清除效率。耐高温菌株在受到高温胁迫后的氧化损伤及氧化修复效果与对照菌株不同,一方面体现在正常状态下维持较低的细胞能量代谢和较高的ROS合成量;另一方面抗氧化系统中sod1、sod2、cat1与对照菌株相比有不同程度的上调,SOD和过氧化氢酶(CAT)活性增强,可以更有效地清除过量的活性氧,减轻高温对菌丝的氧化损伤。尤其在高温胁迫120min时,A15的线粒体功能及抗氧化系统受到严重损伤,线粒体复合物Ⅰ、Ⅱ、Ⅲ活性和CAT活性大幅度下降,但是A15-TH线粒体复合体Ⅰ、Ⅲ活性分别增加至正常状态下的1.4倍和8.9倍,CAT活性比对照菌株高128%,维持了正常的线粒体功能及对活性氧的有效清除。进一步研究发现高温胁迫下,双孢蘑菇菌丝的己糖激酶和丙酮酸激酶活性增加,糖酵解途径加快;耐高温菌株A15-TH在正常状态下和高温胁迫下,己糖激酶和丙酮酸激酶的活性均高于对照菌株A15,具有更活跃的碳代谢。

Industrialized cultivated strain A15 assigned as experimental control and thermotolerant strain A15-TH screened from A15 were used to study the effects of high temperature stress on the growth of mycelia of Agaricus bisporus, and the response to heat and thermotolerant mechanism were explored on the basis of oxidative damage repair and basic carbon metabolism-glycolysis. It was found that under high temperature stress, the growth rate of mycelia of A15 decreased, and hyphal bifurcation increased. However, the growth rate of mycelia of A15-TH was higher than that of A15 and the hyphal morphology was normal and superior to that of A15, manifesting A15-TH was tolerant to high temperature stress. The oxidative damage and antioxidant enzyme system of the two strains were compared under high temperature stress. The results showed that the ATP content reduced by 54.4%–59.6%, the activities of mitochondrial complex Ⅰ, Ⅱ and Ⅲ increased and the superoxide anion(O2-) content increased by 34.9%–71.3% under 30–90 min heat stress for A15. In addition, high-temperature stress reduced the superoxide dismutase activity, thus affected the clearance efficiency of O2-of A15. A15-TH showed low energy metabolism and high O2-synthesis under normal conditions and the expressions of sod1, sod2, and cat1 genes were up-regulated as compared with the control strain A15 under high temperature stress. Moreover, the activities of superoxide dismutase and catalase enhanced, which could remove excess O2-more effectively and reduced the oxidative damage to mycelia. Under 120 min heat stress, the mitochondrial function and antioxidant system of A15 were severely damaged and the activities of mitochondrial complex Ⅰ, Ⅱ, Ⅲ and CAT decreased significantly. On the contrary, under such a heat stress, A15-TH showed normal mitochondrial function and efficient removal of excess O2-, manifesting that mitochondrial complex Ⅰ and Ⅲ activities increased to 1.4 and 8.9 times respectively as compared with the normal state while CAT activity was 128% higher than that of A15. Under high temperature stress, the activity of hexokinase and pyruvate kinase increased, and the glycolytic pathway accelerated. In comparison with A15, A15-TH showed higher activity of hexokinase and pyruvate kinase under both normal condition and high temperature stress, indicating more active carbon metabolism.