真菌侵染诱导沉香形成关键技术效果评价及结香机制初步研究
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摘要
沉香是瑞香科植物白木香(Aquilaria sinensis (Lour.)Gilg)含树脂的木材,是我国、印度及其他东南亚国家的传统名贵中药,具有行气止痛、纳气平喘、温中止呕的功效。同时,其具有浓郁而独特的香气,故常被用来制造高级香水、高级香皂、高级香精等。白木香是国产沉香的唯一植物资源,其树干在受到损伤或刺激的情况下才能分泌沉香树脂,但这需要十几年甚至几十年的累积。天然沉香的偶然性及长周期性已远不能满足市场需求,人们便开始使用各种人工促香方法促进白木香结香并且从未间断对沉香形成机制的探索。
黑色树脂形成于沉香属植物的心材部位,为了找出含有树脂的木材需要砍倒树干,导致大量白木香树遭人为砍伐。白木香本身因木材松软既没有药用价值也无法当建材用,砍伐的白木香树及其叶、果皮、种子等均被作为废物处理。本实验对白木香叶、果皮、种子以及结香部位的化学成分进行了全面分析,在白木香果皮中首次检出大量沉香的特征性成分:芳香族化合物、倍半萜类和色酮类,发现白木香果皮提取物表现出良好的抗肿瘤活性。这为全面合理开发利用白木香植物资源奠定了研究基础。
虽然白木香结香机制尚未明确,但普遍认为与其内生真菌有着密切关系。本课题组使用新的“生物法”—无创伤的“小孔滴注法”。无创伤方法既可排除白木香树干组织受到大面积的物理伤害,又能避免外源环境微生物对树干组织的侵染,从而保障了真菌对沉香诱导作用研究结果的可靠性。为了使“小孔滴注法”人工接种沉香既能实现快速结香又能让其品质达到药典的要求,运用GC-MS分析技术和分子生物学技术研究白木香结香过程中化学成分和结香前后内生真菌群落构成的动态变化,得出一定的动态变化规律并筛选出可快速诱导沉香形成的优势菌株;同时,建立了测定沉香中苄基丙酮含量的气相色谱法,为沉香质量全面评价体系奠定基础。
镰刀菌属于子囊菌亚门,它是大部分白木香分离培养研究中均获得的常见内生真菌,我们也从白木香中分离到了,因此选择镰刀菌进行人工造香试验。建立了“小孔滴注法”人工造香21个月白木香叶内生真菌rDNA ITS区克隆文库,共获得28条序列,分属刺盘孢属、拟盘多毛孢属、拟茎点霉菌、球座菌属、曲霉属、核盘霉属、子囊菌等。其中刺盘孢属、拟盘多毛孢属、拟茎点霉菌为较优势菌,分别占文库的35.7%、10.7%、7.14%。而健康白木香(未结香)叶内生真菌rDNA ITS区克隆文库,得到10条序列,分属刺盘孢属、球腔菌属、枝氯霉属等。二者均具有的内生真菌群落只有刺盘孢属(Collectrotrichum),说明白木香人工结香前后内生真菌群落构成差异较大。在白木香树内接种镰刀菌(Fusarium sp.)后,可能改变其内生真菌的微生态环境,从而导致其内生真菌群落构成发生变化。
Chinese eaglewood is the fragrant wood of Aquilaria sinensis (Lour.)Gilg. (Thymelaeaceae). Eaglewood has been used as traditional antiemetic, analgesic, digestive and tonic in China, India and other southeast Asian countries. Meanwhile, it often used to make advanced perfume, senior soap, senior essence etc. Aquilaria sinensis is the unique resource of Chinese eaglewood in China. Eaglewood produced by damaged or stimulated, these naturally formed resin generally cost more than thirty years. For meeting the demand of market, many artificial methods were used for inducing the oleoresin. And never stopped to explore the mechanism of the eaglewood formation.
Black resins formed in the heartwood parts of eaglewood, people need to cut down trunk in order to find out the resin, which lead to a large number of trees were cut down. It is useless because of the softness of the wood, Aquilaria sinensis itself and its leaves, peel, and seeds were treated as wastes. The experiment is fully analyaed the chemical composition of the leaf, peel, seeds and edgeworthia site. A large number of characteristic elements of A. sinensis were detected for the first time: The chloroform extract from peel of A. sinensis performed significantly antitumor activity. These lay a new foundation of reasonable exploration and utilization of A. sinensis resources.
As far, the mechanism of the eaglewood formation is still an enigma although some fungi were considered closely associate with the formation of eaglewood. The study adopted a new“biological method”–atraumatic“Holes drip method”, which can both exclude tissue suffer from physical damage and avoid infection from exogenous environmental microorganism. The dynamic changes on chemical composition of resins formed process and endophytic fungi by using GC-MS analysis technique and molacular biology technique have abtained dynamic regularity and screened predominant endophyte. Concurrently, we have eatablished an GC method for determing the content of benzylactone, then laid a foundation of comprehensive evalution system for eaglewood quality.
We choose Fusarium sp. for making artificial resins formed test, which belong to Ascomycotion and easily abtained from A. sinensis. Diversity of endophytic fungi from leaves of inoculated A. sinensis was studied by sequencing of the internal transcribed space (ITS) region of fungal ribosomal DNA (rDNA). Twenty-eight sequences were obtained and identified as Collectrotrichum sp., Pestalotiopsis sp., Phomopsis sp., Guignardia sp., Aspergillus sp., Sclerotinia sp., Ascomycete sp., etc. Collectrotrichum sp., Pestalotiopsis sp. and Phomopsis sp.were the dominant species which respectively accounting 35.7%、10.7%、7.14% of all. While ten sequences were obtained from leaves of A. sinensis and identified as Collectrotrichum sp., Mycosphaerella sp., Ramichloridium sp.,etc. Both of the endophytic fungi community has only Collectrotrichum sp., which shows remarkable difference between before and after the forming of resins in endophytic fungi composition. The results infer that it had broken original ecology environment after inoculate Fusarium sp., and then come into being more new kinds of endophytic fungi group.
黑色树脂形成于沉香属植物的心材部位,为了找出含有树脂的木材需要砍倒树干,导致大量白木香树遭人为砍伐。白木香本身因木材松软既没有药用价值也无法当建材用,砍伐的白木香树及其叶、果皮、种子等均被作为废物处理。本实验对白木香叶、果皮、种子以及结香部位的化学成分进行了全面分析,在白木香果皮中首次检出大量沉香的特征性成分:芳香族化合物、倍半萜类和色酮类,发现白木香果皮提取物表现出良好的抗肿瘤活性。这为全面合理开发利用白木香植物资源奠定了研究基础。
虽然白木香结香机制尚未明确,但普遍认为与其内生真菌有着密切关系。本课题组使用新的“生物法”—无创伤的“小孔滴注法”。无创伤方法既可排除白木香树干组织受到大面积的物理伤害,又能避免外源环境微生物对树干组织的侵染,从而保障了真菌对沉香诱导作用研究结果的可靠性。为了使“小孔滴注法”人工接种沉香既能实现快速结香又能让其品质达到药典的要求,运用GC-MS分析技术和分子生物学技术研究白木香结香过程中化学成分和结香前后内生真菌群落构成的动态变化,得出一定的动态变化规律并筛选出可快速诱导沉香形成的优势菌株;同时,建立了测定沉香中苄基丙酮含量的气相色谱法,为沉香质量全面评价体系奠定基础。
镰刀菌属于子囊菌亚门,它是大部分白木香分离培养研究中均获得的常见内生真菌,我们也从白木香中分离到了,因此选择镰刀菌进行人工造香试验。建立了“小孔滴注法”人工造香21个月白木香叶内生真菌rDNA ITS区克隆文库,共获得28条序列,分属刺盘孢属、拟盘多毛孢属、拟茎点霉菌、球座菌属、曲霉属、核盘霉属、子囊菌等。其中刺盘孢属、拟盘多毛孢属、拟茎点霉菌为较优势菌,分别占文库的35.7%、10.7%、7.14%。而健康白木香(未结香)叶内生真菌rDNA ITS区克隆文库,得到10条序列,分属刺盘孢属、球腔菌属、枝氯霉属等。二者均具有的内生真菌群落只有刺盘孢属(Collectrotrichum),说明白木香人工结香前后内生真菌群落构成差异较大。在白木香树内接种镰刀菌(Fusarium sp.)后,可能改变其内生真菌的微生态环境,从而导致其内生真菌群落构成发生变化。
Chinese eaglewood is the fragrant wood of Aquilaria sinensis (Lour.)Gilg. (Thymelaeaceae). Eaglewood has been used as traditional antiemetic, analgesic, digestive and tonic in China, India and other southeast Asian countries. Meanwhile, it often used to make advanced perfume, senior soap, senior essence etc. Aquilaria sinensis is the unique resource of Chinese eaglewood in China. Eaglewood produced by damaged or stimulated, these naturally formed resin generally cost more than thirty years. For meeting the demand of market, many artificial methods were used for inducing the oleoresin. And never stopped to explore the mechanism of the eaglewood formation.
Black resins formed in the heartwood parts of eaglewood, people need to cut down trunk in order to find out the resin, which lead to a large number of trees were cut down. It is useless because of the softness of the wood, Aquilaria sinensis itself and its leaves, peel, and seeds were treated as wastes. The experiment is fully analyaed the chemical composition of the leaf, peel, seeds and edgeworthia site. A large number of characteristic elements of A. sinensis were detected for the first time: The chloroform extract from peel of A. sinensis performed significantly antitumor activity. These lay a new foundation of reasonable exploration and utilization of A. sinensis resources.
As far, the mechanism of the eaglewood formation is still an enigma although some fungi were considered closely associate with the formation of eaglewood. The study adopted a new“biological method”–atraumatic“Holes drip method”, which can both exclude tissue suffer from physical damage and avoid infection from exogenous environmental microorganism. The dynamic changes on chemical composition of resins formed process and endophytic fungi by using GC-MS analysis technique and molacular biology technique have abtained dynamic regularity and screened predominant endophyte. Concurrently, we have eatablished an GC method for determing the content of benzylactone, then laid a foundation of comprehensive evalution system for eaglewood quality.
We choose Fusarium sp. for making artificial resins formed test, which belong to Ascomycotion and easily abtained from A. sinensis. Diversity of endophytic fungi from leaves of inoculated A. sinensis was studied by sequencing of the internal transcribed space (ITS) region of fungal ribosomal DNA (rDNA). Twenty-eight sequences were obtained and identified as Collectrotrichum sp., Pestalotiopsis sp., Phomopsis sp., Guignardia sp., Aspergillus sp., Sclerotinia sp., Ascomycete sp., etc. Collectrotrichum sp., Pestalotiopsis sp. and Phomopsis sp.were the dominant species which respectively accounting 35.7%、10.7%、7.14% of all. While ten sequences were obtained from leaves of A. sinensis and identified as Collectrotrichum sp., Mycosphaerella sp., Ramichloridium sp.,etc. Both of the endophytic fungi community has only Collectrotrichum sp., which shows remarkable difference between before and after the forming of resins in endophytic fungi composition. The results infer that it had broken original ecology environment after inoculate Fusarium sp., and then come into being more new kinds of endophytic fungi group.
引文
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