【作者】 曹晋静；

【导师】 康冀川；

【作者基本信息】 贵州大学 ， 微生物学， 2009， 硕士

【摘要】 微生物生态学是研究微生物群体与其周围生物和非生物环境条件间相互作用规律的科学。植物微生态学是研究植物体内微生物的组成、功能、演替,它们互相之间关系及其与寄主间相互关系的生命科学分支。植物内生真菌(Endophytic fungi)是指在某一时期生活在健康植物体内但对寄主植物组织并不引起明显侵染的真菌。某些内生真菌能产生与宿主相似的活性代谢产物而成为新药的来源之一。内生真菌数量庞大,其作为一种新型的微生物资源,已成为人们关注的热点。本文以我国特有药用植物——喜树(Camptothecaa cuminata Decne.)为实验材料,对贵州大学校园内不同季节的喜树的根、茎、叶、种子进行内生真菌的分离,共分离得到菌株151株。并且还分离了喜树周边环境如根际土壤、空气、雨水中的真菌,在所试条件下分别得到菌株72株、60株、53株,采用形态学和分子生物学结合的方法对所得菌株进行了鉴定。采用TLC法和HPLC法从151株内生真菌中共筛选到4株能产生喜树碱及其类似物的内生真菌,实验结果如下：1、对其中70株内生真菌进行了鉴定,鉴定为15个属,11个科,10个目,5个纲,分属3个亚门。鉴定结果在一定程度上反应了喜树内生真菌的生物多样性以及季节差异性,并且发现拟茎点霉属(Phomopsis)是喜树内生真菌的优势菌群。对部分土壤、空气和雨水真菌也做了鉴定,结果为：土壤真菌分属于3个亚门,4个纲,7个目,8个科,13个属；空气真菌分属3个亚门,4个纲,7个目,7个科,10个属；雨水真菌分属3个亚门,3个纲,7个目,7个科,9个属。2、从分离鉴定结果中发现,除了拟茎点霉属(Phomopsis)以外,链格孢属(Alternaria)和镰孢属(Fusarium)也是喜树内生真菌中分离比较多的菌属,而这两种菌属在环境中也很容易分离得到。将环境中的真菌与内生真菌相比我们可以看出,从植物体内分离到的内生真菌,15个属中除了葡萄座腔菌属(Botryosphaeria)、丝核菌属(Rhizoctonia)、黑孢霉属(Nigrospora)、腐皮壳属(Diaporthe)和枝顶孢霉属(Acremonium)这5个属以外,其余的属在环境中都有存在,只是数量上不同而已。同时,有的环境真菌在植物体内又不一定有,例如：木霉属(Trichoderma)、拟青霉属(Paecilomyce)、酵母菌(yeast)、轮枝霉属(Verticillium)、裂褶菌属(Schizophyllum)和显革菌属(Phanerochaete)的白腐菌(Trametes versicolor)。这在一定程度上反映了喜树内生真菌与环境真菌之间存在着密切的关系。3、本实验使用PD培养基对151株内生真菌进行发酵时,未筛选出产喜树碱及其类似物的内生真菌,而采用喜树种子煎汁作为诱导物进行诱导培养,即在PD培养基中加入了喜树种子的煎汁,结果从151株内生真菌中共筛选到4株能产生喜树碱及其类似物的内生真菌,其中产喜树碱的3株,产10-羟基喜树碱的1株。说明了这4株喜树内生真菌是在喜树种子产生的某种前体物质刺激下产生了喜树碱及其类似物。经形态学和分子生物学的鉴定,这4株菌分别为QZ04：链格孢属(Alternaria sp.)、CY02:拟茎点霉属(Phomopsis sp.)、XY09:镰孢属(Fusarium sp.)、XY05:刺盘孢属(Colletotrichum sp.),含量分别是0.029mg/g、0.024mg/g、0.037mg/g、0.017mg/g。随着人们对喜树碱类抗癌药物需求的增加,单纯靠从喜树中提取喜树碱会造成资源的短缺和生态环境的破坏,本实验筛选出了能产喜树碱及其类似物的内生真菌,为喜树碱的开发寻找新的途径提供了一定的理论依据和参考价值。更多还原

【Abstract】 Microbial Ecology is the study of microbial groups and their surrounding environmental conditions and their interaction. Plant microecology is the study of microbial composition, function, succession and their mutual relations and interactions with the host plant.Endophytic fungi are fungi that colonize the inside of healthy plant tissues, persisting for a certain period of time without causing visible disease symptoms. Some endophytic fungi can produce bioactive metabolites, similar to their host, and become a novel source of potentially useful medicinal compounds. Endophytic fungi, which vastly exist in nature, have been a research focus in recent years and they become new sources of microorganism.In this thesis, Camptotheca acuminata Decne., a chinese medicinal plant, was selected as the ecological niche for the studies. A total of 151 endophytic fungal isolates from the roots, stems, leaves, seeds of C. acuminata Decne. at Guizhou University campus on the different seasons were isolated. Additionally 72,60 and 53 fungal isolates were isolated from the environmental soil, air and rain respectively. Thin layer chromatography(TLC) method and the high effective liquid chromatography(HPLC) were employed to detect metabolites. The results indicated that the mycelia of 4 isolates contained camptothecine,10-hydroxycamptothecine and similar compounds. The results were as follows:1. The research investigates endophytic fungi diversity in C. acuminata Decne. and 70 isolates of endophytic fungi were isolated. The isolates were identified to belong to 15 genera,11 families,10 orders,5 class and 3 phylum with morphological and molecular means. The biodiversity and seasonal differences of endophytic fungi in C. acuminata were studied and the dominant groups were Phomopsis spp. Fungal isolates were isolated and identified from the soil, air and rain. The results were as follows:the isolates of soil belong to 13 genera,8 families,7 orders,4 class and 3 phylum; the isolates of air belong to 10 genera,7 families,7 orders,4 classes and 3 phylum and the isolates of rain belong to 9 genera,7 families,7 orders,3 classes and 3 phylum.2. The results showed that in addition to Phomopsis, Alternaria and Fusarium were also easily isolated. from C. acuminata Decne. and the environment. Apart from Botryosphaeria, Rhizoctonia, Nigrospora, Diaporthe, and Acremonium, the other fungal genera of the 15 endophytic fungi genera exist in the environment. While Trichoderma, Paecilomyce, yeast, Verticillium, Schizophyllum and Trametes versicolor isolated from the environment do not exist in the host plant. These results reflected the relationship of endophyte fungi and the fungi in the environment.3. With ordinary PD media, no camptothecin and similar compounds were found in the 151 endophytic fungi isolates. With the addition of the seed decocting liquid t o PD media, camptothecin and similar compounds were detected in 4 isolates from the 151 endophytic fungi. With morphological and molecular means, these 4 isolates QZ04, CY02, XY09, XY05 were identified as Alternaria sp., Phomopsis sp., Fusarium sp. and Colletotrichum sp. respectively. The contents of them were 0.029mg/g,0.024mg/g,0.037mg/g and 0.017mg/g respectively.With increasing demand for anti-cancer drug camptothecin, extraction of camptothecin from C. acuminata Decne. caused the shortage of resources and ecological damage to the environment. This research screened endophytic fungal isolates to produce camptothecin and similar compounds which could potentially used for the production camptothecin in the future.更多还原