草菇菌种在无性继代培养中的退化机制研究

Volvariella volvacea (Bull.: Fr.) Sing is a kind of nourishing and delicious mushroom, it is ranked the fifth most important edible mushroom in the world in terms of yield. Due to its cellulolytic characteristics, V. volvacea has been extensively cultivated on an array of agricultural residues under artificial conditions. However, V. volvacea is a typical high temperature type edible mushroom with weak low temperature tolerance. Mycelia subculture and fruit body tissue isolation can result in pawn degradation of V. volvacea, which cause yield reduction and serious economic loss. The differentially expressed genes and the metabolic biomarkers of degradation spawn were decteted using mass spectrometry (GC/MS)-based metabolomics and RNA-seq transcriptomics in this project. Meanwhile, cellulase activity, production quota and fruit body characters of degradation strains will be determined. Implementation of this project will reveal spawn degeneration phenomenon during mitotic subculture of V. volvacea, and provide comprehensive information of molecular mechanisms and metabolic characters to degradation of V. volvacea, The project will also offer the theory basis of early identification and control technologies through modern biotechnology to degradation of V. volvacea.

草菇是世界产量第五的食用菌，味道鲜美，营养丰富，同时具有较强的纤维素分解能力，能消耗大量的农业废料。草菇是典型的高温型食用菌，不耐低温储藏，在菌丝体继代和子实体组织分离继代过程中，都会产生菌种退化现象，使得草菇产量下降，造成严重的经济损失。本项目结合两种组学对无性继代下的草菇退化菌株进行代谢组学（GC-MS）和转录组学（RNA-seq）分析，筛选与退化有关的差异表达基因和代谢标志物。同时测定草菇退化菌株的纤维素酶活变化规律和规模化栽培试验中的草菇生产性状和子实体指标。本项目的开展可望全面了解草菇菌种在无性继代过程中发生的退化现象，并从分子机制和代谢调控上阐明草菇菌种在继代过程中的退化机理，为草菇菌种退化的早期鉴定及利用现代生物技术控制菌种退化提供理论依据。

连续菌丝体或组织分离继代均会导致草菇菌种退化，造成严重的经济损失。本项目利用生理生化、组学等方法对草菇菌种退化现象和退化机制进行研究，主要结果如下：（1）尖端菌丝连续继代20个月获得S1-S20；子实体组织分离连续继代16次获得M1-M16，对照为S0和M0。随着继代次数增加，两组继代菌株的菌丝生长速度和生物量均先增加后降低。栽培试验表明：S0-S12和M0-M16可正常出菇，S13-S20无法出菇。S0-S12的生产周期逐渐延长，生物学效率逐渐下降；M0-M16为先缩短再延长，先升高再降低。LBL染色法可有效鉴定出草菇退化菌种。（2）S0-S12的子实体多糖、蛋白质、黄酮、多酚、矿质元素含量逐渐下降；总氨基酸含量差异不显著，必需氨基酸含量下降；滤纸酶、内切葡聚糖酶、ß-葡萄糖苷酶、木聚糖酶、漆酶、多酚氧化酶和锰过氧化物酶活性均逐渐降低。M0-M16子实体营养成分均先上升后下降；各酶活力均先增加后降低，M4最高。（3）对S0、S4、S8、S12、S16、S20及M0、M4、M8、M12、M16进行转录组学分析。GO功能显示，差异表达基因主要富集在生物粘附、代谢过程和催化活性上。KEGG pathway显示差异表达基因主要在氨基酸代谢、碳水化合物代谢、次生代谢产物合成与分解代谢上。与基质降解和活性氧清除有关的差异表达基因数为27和20个。RT-PCR验证与转录组学数据一致。代谢组学分析表明：出菇组含有3种生物标记物，未出菇组含有6种生物标记物。（4）草菇菌种可能的退化机制为：连续无性继代培养造成草菇基质降解和活性氧清除相关基因表达量下调，降低了相关酶的活力，从而抑制了菌体能量的获得及营养物质合成，加剧活性氧对菌种的毒害作用，进一步造成菌体生长受阻，导致菌种退化现象发生。以上结果为草菇菌种退化鉴定及退化机理研究提供技术手段及理论依据。