杜仲内生真菌的分离与拮抗菌株的筛选

发布时间：2018-05-26 13:04

本文选题：杜仲 + 内生真菌　；参考：《河南大学》2015年硕士论文

【摘要】：杜仲(Eucommia ulmoides Oliv.)为杜仲科杜仲属落叶乔木,中国特有经济树种,国家二级保护植物。其次生代谢产物如绿原酸、桃叶珊瑚甙、京尼平甙等具有抗菌、抗肿瘤等药理活性。植物内生菌普遍存在于各种植物中,许多内生菌能产生与宿主植物相同或相似的代谢产物,且许多是未发现过的新物质。利用内生菌筛选生物活性成分或先导化合物成为寻找天然药物的又一条重要途径,并且许多化合物对动植物致病菌有明显的抑制作用,因此在医药领域和农药领域,植物内生菌是目前研究的热点。同一种植物生长在不同的环境,其内生菌的多样性及生物活性都有所不同。目前,杜仲内生真菌研究主要集中于陕西、四川、上海等地,更重要的是缺乏对分离内生真菌活性成分的研究。河南省开封市紧临黄河,位于华中与华北地区分界点,其独特的地理位置及气候条件为研究筛选植物内生菌提供了独一无二的天然资源。本研究采用组织分离法从生长于河南大学药用植物园的杜仲叶和果实分离内生真菌,通过观察菌落性状、大小、颜色筛选。培养液用高效液相色谱法(HPLC)检测培养液中是否含有绿原酸、桃叶珊瑚苷等杜仲的主要活性成分。对峙培养评价每个菌株的抗菌活性。对于对峙法有抗菌活性的菌株,用生长速率法评价抗菌活性。对于抑菌效果好的内生真菌,测定ITS片段,Blast比对鉴定其种类。希望能从中分离到具有良好抑菌效果的内生真菌,为进一步提取优良生物活性和新颖化学结构的内生真菌代谢产物打下基础。结果如下:1.菌种分离用1/4划线法对长出的杜仲内生菌进行分离纯化,用形态观察法和显微检测法对杜仲内生菌菌丝和孢子进行检测分类,结果显示:一共分出91株形态不同的菌株。2.杜仲内生菌次级代谢产物化学成分测定7个菌株能够产生桃叶珊瑚苷;2个菌株能够产生绿原酸;没有菌株能够产生黄酮。3.杜仲内生菌抑菌实验分别采用对峙法和菌丝生长速率法对杜仲内生菌进行抑菌活性的检测,供试菌禾谷丝核菌(Rhizoctonia cerealis),小麦根腐(Drechslera Sofokiniana),小麦赤霉病禾谷镰孢(Fusarium graminearum),蜡样芽孢杆菌(Bacillus cereus);人类致病菌有:金黄色葡萄球菌(Staphylococcus aureus),白色念珠菌(Monilia albican),猪霍乱沙门氏菌(Salmonella choleraesuis),大肠杆菌(Escherichia coli)。由抑菌实验结果可以看出分离出的菌株中对植物源的供试菌具有抑制作用的菌株较多,且对植物源供试菌具有较好的抑制作用;对动物源的供试菌具有抑制作用的菌株较少,且对动物源供试菌抑制作用较差。4.分子鉴定:用试剂盒提取杜仲内生菌DNA,进行PCR扩增,生物公司测序后,序列在Gene Bank上进行blast比对,对内生菌进行分子学鉴定,鉴定结果显示:1、2、7、9、11、16、18、23、26、27、29、30、42、46号菌株同属于链格孢属(Alternaria);41号菌株属于茎点霉属(Phoma);4号菌株属于黑孢属(Niqrospora);31、49号菌同属于枝孢属(Cladosporium)。
[Abstract]:Eucommia ulmoides Oliv. (Eucommia ulmoides) is a deciduous tree of Eucommia ulmoides (Eucommia ulmoides), a special economic tree in China and a national two level protection plant. The secondary metabolites, such as chlorogenic acid, glucoside, geniposide, etc., have antibacterial and anti-tumor pharmacological activities. Endophytes are commonly found in various plants, and many endophytes can produce and host the host Plants are the same or similar metabolites, and many are undiscovered new substances. Using endophytes to screen bioactive ingredients or precursor compounds is another important way to find natural drugs, and many compounds have obvious inhibitory effects on animal and plant pathogens. Therefore, plant endophytes in the field of medicine and pesticides are endophytic bacteria. At present, the study of endophytic fungi in Eucommia ulmoides is mainly concentrated in Shaanxi, Sichuan, Shanghai and other places, and more important is the lack of research on the activity of isolated endophytic fungi. In Kaifeng, Henan Province, the Yellow River, China is located in China. The unique geographical location and climate conditions provide unique natural resources for the study and screening of endophytic bacteria. This study uses tissue separation method to isolate endophytic fungi from the leaves and fruits of Eucommia ulmoides from the medicinal botanical garden of Henan University. The main active components of Eucommia ulmoides, such as chlorogenic acid and coronoside, were detected by high performance liquid chromatography (HPLC). The antibacterial activity of each strain was evaluated by confrontation culture. The antibacterial activity was evaluated by the growth rate method. For the endophytic fungi with good bacteriostasis effect, ITS fragments and Blast were measured. It is hoped to separate the endophytic fungi with good bacteriostasis effect and lay the foundation for the further extraction of endophytic metabolites with excellent biological activity and novel chemical structure. The results are as follows: 1. the isolation and purification of Endophytes in Eucommia ulmoides by 1/4 line separation method by the method of morphological observation and microscopy Detection and classification of endophytic mycelium and spores of Eucommia ulmoides, the results showed that 91 strains of different strains of Eucommia.2. were identified by the chemical composition of secondary metabolites of Eucommia ulmoides (Eucommia ulmoides), 7 strains could produce anthocyanin, and 2 strains could produce chlorogenic acid; no strains could produce bacteriostasis experiment of flavonoids of Eucommia ulmoides.3. Do not use confrontation and mycelium growth rate method to detect the Bacteriostasis of Eucommia endophyte, for Rhizoctonia cerealis, wheat root rot (Drechslera Sofokiniana), wheat scab (Fusarium graminearum), and Bacillus cereus (Bacillus cereus), and Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus), Candida albicans (Monilia albican), swine cholera Salmonella (Salmonella choleraesuis) and Escherichia coli (Escherichia coli). From the results of bacteriostasis experiments, we can see that the isolated strains have more inhibitory effects on plant source bacteria, and have a better inhibition effect on plant source bacteria. The strains with inhibitory effects on the source of the animal source were less, and the inhibition effect of the animal source for the test bacteria was poor.4. molecular identification: the DNA of Eucommia ulmoides ulmoides was extracted with the kit, and the PCR amplification was carried out. After the sequencing of the biological company, the sequence on the Gene Bank was carried out by blast comparison, and the endophyte was identified by the molecular identification. The identification results showed that 1,2,7,9,11,16,18 The strain 23,26,27,29,30,42,46 belongs to the genus Alternaria; the strain 41 belongs to the genus Phoma; the strain 4 belongs to the genus Niqrospora; 31,49 is belong to the genus Cladosporium (Cladosporium).
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S567.19;S182

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