皱边石杉内生菌的分离、纯化及种属初步鉴定
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摘要
目前,石杉碱甲这一治疗阿尔茨海默病(Alzheimer's disease, AD)的特效药,主要从石杉科植物中提取,由于其在植物体含量很低,这些植物又属于原始蕨类植物,生长缓慢,生长周期长,采摘后植株无法再生,为一次性利用资源。加上野生资源的日益减少,人工栽培又非常困难。而国内外学者对它的全合成、衍生物及类似物的合成作了大量的工作,但至今几乎没有得到活性比天然Hup A更好的衍生物和类似物,寻找到新的Hup A的合成途径就非常有意义。
由于伴有真菌共生的原因,很难解决材料组织培养的污染问题,所以皱边石杉这类低等且伴有内源真菌的蕨类植物组织培养实验没有取得突破性进展。目前国内外研究多数集中在石杉碱甲的临床药理作用上,对皱边石杉及其内生菌的研究工作开展很少。
根据内共生理论,内生真菌可能产生与宿主相同或相似的次生代谢产物。已有研究发现,药用植物的内生真菌可产生与中药相同或相似的化合物。所以,药用植物内生真菌一直是近年来筛选一些重要医用药物的研究热点,特别是生产成本高、价格昂贵或其它途径不易得到的药物,极具发展前景。
本论文在前期预实验的基础上,发现皱边石杉组织培养外植体存在内生菌,根据前人对内共生真菌与宿主的协同进化机理,进行了皱边石杉内生菌的研究。以皱边石杉新鲜植物为材料,采用MS培养基进行组织培养,从组织培养材料的二次消毒材料中初步分离内生菌,并用马铃薯固体培养基(PDA)对皱边石杉内生菌进行了多次分离、纯化,得到72原始菌株。菌种在石蜡油中长期保存,PDA改良培养基进行菌种复兴、PDA常规培养基进行菌种菌落形态学观察、斜插法制片观察菌丝体孢子体形态。在培养过程中仔细记录其形态学特征(菌落大小、颜色、表面纹饰质地、高度、边缘、渗出物)及个体特征(菌丝有无隔膜,有无细胞核,分生孢子梗分枝情况及分生孢子的形态),参考工具书,对所获得的内生真菌进行形态学的初步鉴定。为后期进行功能菌株的发酵、功能产物的鉴定提供菌种材料。本研究将其中53种菌株鉴定到属,分别是青霉属、曲霉属、无孢类群、根霉属、红酵母属、交链孢霉属。其中,青霉属和曲霉属是优势菌株。
Huperzine A (Hup A) is a specific medicine for Azheimer's disease (AD), mainly extracting from the Huperziaceae. Low natural contents, long growth cycle and non-regeneration after picked plus rare natural source and difficulties in artificial cultivation lead to the lack of natural Hup A. Many works have been done in researching its compounds, ramifications, analogues but failed to find better alternatives in activity. Thus, it's significant of finding a biosynthetic way of Hup A.
Because the contamination of Entophytic fungus usually causes problems in doing tissue culture experiments of Huperzia crispata (Ching) and such lower plants in the family, most research so far focused on the pharmacology effect and clinical application of Hup A and little attention was paid on the study of Huperzia criapata and its endophytic fungus.
The theory of endosymbiosis shows that endophytic fungus might produce the same or similar secondary metabolites. Based on the theory, endophytic fungus of medical herbs might produce compounds which could function effectively. Recent studies found various beneficial secondary metabolites and thus made it easier to screen new compounds. It brought a new method to overcome high production cost and sourcing problems.
On the basis of preparative experiment, we found there are endophytic fungus in the explant of Huperzia criapata, and according to co-evolution mechanism of symbiotic fungus and endophytic fungus, this article mainly focused on endophytic fungus of Huperzia criapata. The author uses fresh Huperzia crispata (Ching) as material, preliminarily isolated fungus from cultivated materials of Huperzia criapata on MS medium,Then didi severals seperations and purifications on PDA, we get 72 original fungus of Huperzia criapata. The fungs stores in the paraffin oil.Using developed PDA acticvate the fungus,and observing the morphologies. Record the morphologic Characteristics, including the diameter,the color, the ornamentation, texture, hight, theedge, exudation), and individual characteristics (septum, Nucleolus, conidiophore). According to reference books,the author did preliminary identification of the fungus, providing samples for the fermention of function fungus and identifying functional products. Preliminary Identification 53 fungus,including Penicillium Lk.ex.Fr., Aspergillus Mich.ex Fr., Mycelia Sterillia, Rhizopus Ehrenberg, Rhodotorula Harrison, Alternaria Nees ex Wall. The Penicillium Lk.ex Fr. and the fungus of Aspergillus Mich.ex Fr. are the dominant.
由于伴有真菌共生的原因,很难解决材料组织培养的污染问题,所以皱边石杉这类低等且伴有内源真菌的蕨类植物组织培养实验没有取得突破性进展。目前国内外研究多数集中在石杉碱甲的临床药理作用上,对皱边石杉及其内生菌的研究工作开展很少。
根据内共生理论,内生真菌可能产生与宿主相同或相似的次生代谢产物。已有研究发现,药用植物的内生真菌可产生与中药相同或相似的化合物。所以,药用植物内生真菌一直是近年来筛选一些重要医用药物的研究热点,特别是生产成本高、价格昂贵或其它途径不易得到的药物,极具发展前景。
本论文在前期预实验的基础上,发现皱边石杉组织培养外植体存在内生菌,根据前人对内共生真菌与宿主的协同进化机理,进行了皱边石杉内生菌的研究。以皱边石杉新鲜植物为材料,采用MS培养基进行组织培养,从组织培养材料的二次消毒材料中初步分离内生菌,并用马铃薯固体培养基(PDA)对皱边石杉内生菌进行了多次分离、纯化,得到72原始菌株。菌种在石蜡油中长期保存,PDA改良培养基进行菌种复兴、PDA常规培养基进行菌种菌落形态学观察、斜插法制片观察菌丝体孢子体形态。在培养过程中仔细记录其形态学特征(菌落大小、颜色、表面纹饰质地、高度、边缘、渗出物)及个体特征(菌丝有无隔膜,有无细胞核,分生孢子梗分枝情况及分生孢子的形态),参考工具书,对所获得的内生真菌进行形态学的初步鉴定。为后期进行功能菌株的发酵、功能产物的鉴定提供菌种材料。本研究将其中53种菌株鉴定到属,分别是青霉属、曲霉属、无孢类群、根霉属、红酵母属、交链孢霉属。其中,青霉属和曲霉属是优势菌株。
Huperzine A (Hup A) is a specific medicine for Azheimer's disease (AD), mainly extracting from the Huperziaceae. Low natural contents, long growth cycle and non-regeneration after picked plus rare natural source and difficulties in artificial cultivation lead to the lack of natural Hup A. Many works have been done in researching its compounds, ramifications, analogues but failed to find better alternatives in activity. Thus, it's significant of finding a biosynthetic way of Hup A.
Because the contamination of Entophytic fungus usually causes problems in doing tissue culture experiments of Huperzia crispata (Ching) and such lower plants in the family, most research so far focused on the pharmacology effect and clinical application of Hup A and little attention was paid on the study of Huperzia criapata and its endophytic fungus.
The theory of endosymbiosis shows that endophytic fungus might produce the same or similar secondary metabolites. Based on the theory, endophytic fungus of medical herbs might produce compounds which could function effectively. Recent studies found various beneficial secondary metabolites and thus made it easier to screen new compounds. It brought a new method to overcome high production cost and sourcing problems.
On the basis of preparative experiment, we found there are endophytic fungus in the explant of Huperzia criapata, and according to co-evolution mechanism of symbiotic fungus and endophytic fungus, this article mainly focused on endophytic fungus of Huperzia criapata. The author uses fresh Huperzia crispata (Ching) as material, preliminarily isolated fungus from cultivated materials of Huperzia criapata on MS medium,Then didi severals seperations and purifications on PDA, we get 72 original fungus of Huperzia criapata. The fungs stores in the paraffin oil.Using developed PDA acticvate the fungus,and observing the morphologies. Record the morphologic Characteristics, including the diameter,the color, the ornamentation, texture, hight, theedge, exudation), and individual characteristics (septum, Nucleolus, conidiophore). According to reference books,the author did preliminary identification of the fungus, providing samples for the fermention of function fungus and identifying functional products. Preliminary Identification 53 fungus,including Penicillium Lk.ex.Fr., Aspergillus Mich.ex Fr., Mycelia Sterillia, Rhizopus Ehrenberg, Rhodotorula Harrison, Alternaria Nees ex Wall. The Penicillium Lk.ex Fr. and the fungus of Aspergillus Mich.ex Fr. are the dominant.
引文
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