高氧处理调控采后灵芝三萜类物质代谢机理的研究

The contents of bioactive compound in Ganoderma lucidum fruiting body drop gradually during the storage, therefore, its quality, health and nutritional benefits decrease. It could not only keep the bioactive compound but also provide a now product for consumer to store fresh fruiting body of G. lucidum with suitable method. Some researches showed high oxygen atmosphere was benefit on the biosynthesis of triterpenes in G. lucidum. G. lucidum fruiting body will be treated by high oxygen atmosphere and the changes of respiratory related enzymes, reactive oxygen species concentration and its generation and scavenging enzymes will be analyzed in this project. The changes of metabolic intermediate, key biosynthesis enzymes and monomer components of triterpenes in G. lucidum fruiting body will also be determined during treatment. The physiological metabolic characterization in G. lucidum under high oxygen atmosphere treatment will be investigated. The relationship between reactive oxygen species and triterpene metabolic will also be elucidated by the results of this project. To insight into the molecular mechanism of high oxygen atmosphere treatment on triterpene biosynthesis and reactive oxygen species metabolism, the difference in gene expression of postharvest G. lucidum will be investigated by transcriptome sequencing, gene functional annotation, classification, metabolic pathways analysis and other bioinformatics technology. The role of differentially expressed genes in reactive oxygen species metabolism and triterpene biosynthesis will be explored and the results will be validated by real time RT-PCR. The possible down-biosynthesis pathway of triterpene in G. lucidum will also be elucidated by these results. The results from this project will elucidate the physiological, biochemical and molecular mechanism, provide fundamental basis for postharvest techniques and quality maintaining of G. lucidum fruiting body and serve as a guide for promoting the development of G. lucidum industry.

灵芝在贮存过程中活性成分含量会逐渐下降，严重影响品质和功效。采用适宜的技术贮藏采后鲜灵芝，既可保持其活性成分，又能为消费者提供新的产品类型。已有研究发现高氧处理有利于灵芝三萜的合成，其调控机制可能与活性氧代谢有关，本项目拟研究高氧条件下灵芝子实体呼吸代谢相关酶、活性氧含量与代谢相关酶以及三萜类物质代谢主要中间产物、关键酶、单体组分组成的变化，揭示灵芝应对高氧处理的生理代谢特征，阐明高氧调控三萜类物质合成与活性氧代谢的关系；对高氧处理的采后灵芝转录组进行测序，生物信息学方法进行基因功能注释、分类和代谢途径分析，筛选与鉴定高氧条件下的差异表达基因，结合实时荧光定量PCR探明其在活性氧代谢、三萜类物质合成与调节中的作用，解析灵芝三萜合成途径的下游环节，从理论上探明高氧处理调控采后灵芝三萜代谢的生理生化机制与分子机理，为采后灵芝的品质保持提供理论基础和技术支撑，以期指导灵芝产业的发展。

灵芝（Ganoderma lucidum）是中国传统的名贵食药用真菌，灵芝三萜是其中的主要活性成分，为了促进采后灵芝子实体中三萜成分的含量，我们采用高氧（60%氧浓度）处理新鲜灵芝子实体，结果表明灵芝呼吸速率会受到抑制，处理前期（第1天），灵芝子实体内过氧化氢（H2O2）和超氧阴离子自由基（O2−∙）含量高于对照组，但随着处理的进行，显著减缓活性氧（ROS）含量，减少丙二醛（MDA）积累，提升超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和琥珀酸脱氢酶（SDH）活性，增加灵芝三萜和酚类含量，其中第3天灵芝三萜含量达5.89 g kg-1（比对照增加了1.13倍），总酚含量达3.69gGAE kg-1（比对照增加了1.31倍），说明一定的环境胁迫压力可以激发灵芝启动自身的抗氧化系统，保护机体免受氧化损伤，并促进相关次生代谢产物的合成。qPCR分析表明，灵芝三萜生物合成关键酶，3-羟基3-甲基戊二酰CoA合成酶(hmgs)、 3-羟基3-甲基戊二酰CoA还原酶(hmgr)、法尼基二磷酸合成酶(fps)、角鲨烯合成酶(sqs) 和环氧角鲨烯环化酶(osc)基因在60%氧气处理的灵芝子实体中均上调，hmgs、hmgr、fps、sqs和osc的mRNA水平分别是对照的2.95-、14.98-、6.18-、7.61-和4.26倍。转录组分析表明，60%氧处理3天后的灵芝子实体有6684个基因表达量上调，3423个基因表达量下调， GO 富集分析显示差异基因主要涉及细胞膜、膜的组成成分、催化活性、氧化还原过程、糖代谢、活性氧代谢等，KEGG 分析显示的差异表达基因主要富集在同源重组、碱基切除修复、错配修复、牛磺酸代谢、抗坏血酸代谢、泛醌和其它萜类生物合成等代谢通路。抗氧化活性分析表明，60%氧处理3天显著提升灵芝子实体样品清除DPPH•、OH•和O2-•的作用，活性分别达到最大值（0.32gGAE kg-1、2.34gTE kg-1和3.32gTE kg-1），为对照组的1.28倍、1.30倍和1.54倍。项目解析了高氧处理调控采后灵芝三萜代谢的生理生化机制与分子机理，为采后灵芝的品质保持提供了基础。