苦马豆内生真菌分离鉴定及其生物碱成分分析

发布时间：2018-11-07 07:43

【摘要】：1.苦马豆内生真菌分离与鉴定为探明苦马豆中是否存在内生真菌及其内生真菌菌落与分布情况,本研究对采自内蒙古阿拉善盟草原的苦马豆样品采用组织分离法对内生真菌进行分离,运用传统形态学与分子生物学方法对所得菌株进行种属鉴定。此外,为了获得较好的分离效果,本次试验也对苦马豆不同组织的最佳消毒时间进行确定,发现苦马豆茎和种子用75%乙醇及2%NaClO最佳表面消毒时间分别为90s、5min,40s、2.5min。本次试验从苦马豆的茎、叶、叶柄和种子进行分离共得到70株内生真菌,其分属于交链孢属(Alternaria Nees)、镰刀孢属(Fusarium sp.)、枝孢属(Cladosporium sp.)、暗梗单孢霉属(Chloridium Lk.)、壳色单隔孢属(Diplodia sp.)、隔孢腔菌属(Pleosporales sp.)、小双胞腔菌(Didymella fabae)、Laetisaria arvalis和Preussia australiis等9个属。并且有3株鉴定到种为Didymella fabae、Laetisaria arvalis和Preussia australiis。Alternaria Nees的相对分离频率最高(61.43%),为苦马豆内生真菌的优势菌属。各样品组织中,叶的分离率(19.58%)最高,种子(41.39%)次之,表明种子和叶是内生真菌侵染和定殖的主要部位。2.苦马豆化学成分系统预试验按照植物化学成分系统预试的常规方法,对苦马豆的化学成分进行了系统预试验。结果表明:苦马豆含有甾体、萜类、挥发油、油脂、糖、有机酸、氨基酸、肽、蛋白质、生物碱、黄酮、香豆素、内酯类化合物、皂苷、鞣质、酚类、强心苷类、蒽醌类。3.苦马豆生物碱TLC分析本试验采用TLC分析、平面色谱图像定量分析软件及柱层析分离等技术,对苦马豆生物碱成分进行分析和分离。结果表明,苦马豆5.00 kg乙醇热回流提取得总浸膏650.20 g,经酸化、碱化,所得碱液依次用酸性氯仿、碱性氯仿、碱性正丁醇萃取,得到三部分浸膏分别为3.01g、0.53g、23.13 g,提取率分别为1.51%、0.27%、11.57%,说明苦马豆生物碱主要集中在正丁醇提取部分,以大极性生物碱为主。对各部分提取物进行薄层层析分析,结果显示碱性氯仿和碱性正丁醇提取部分生物碱至少分别有5种。经与标准样品对照,证明苦马豆所含的生物碱主要以苦马豆素等吲哚里西啶生物碱。
[Abstract]:1. The isolation and identification of endophytic fungi were used to investigate the existence and distribution of endophytic fungi and endophytic fungi. In this study, endophytic fungi were isolated by tissue separation from the samples from Alashan grassland in Inner Mongolia, and the strains were identified by traditional morphological and molecular biological methods. In addition, in order to obtain a better separation effect, the optimum disinfection time of different tissues was also determined. It was found that the optimum surface disinfection time for stems and seeds of Prunus amurensis was 90 s / 5 min and 40 s, respectively, with 75% ethanol and 2%NaClO, respectively. 2.5min. In this experiment, 70 endophytic fungi were isolated from stem, petiole and seed of Sophora spp., belonging to (Alternaria Nees), (Fusarium sp.), (Cladosporium sp.), (Chloridium Lk.),. The genus (Diplodia sp.), belongs to 9 genera of (Pleosporales sp.), (Didymella fabae), Laetisaria arvalis and Preussia australiis. Three strains were identified as Didymella fabae,Laetisaria arvalis and Preussia australiis.Alternaria Nees with the highest relative isolation frequency (61.43%), which was the dominant genus of endophytic fungi. The isolation rate of leaves was the highest (19.58%), followed by seeds (41.39%), indicating that seeds and leaves were the main sites of endophytic fungi infection and colonization. 2. In accordance with the conventional method of plant chemical composition system, the chemical composition of Kudou was tested systematically. The results showed that there were steroids, terpenes, essential oils, sugars, organic acids, amino acids, peptides, proteins, alkaloids, flavonoids, coumarins, lactones, saponins, tannins, phenols, cardiosides, anthraquinones. TLC analysis of alkaloids in Kudou beans by means of TLC analysis, quantitative analysis software of plane chromatographic image and column chromatography, the alkaloids were analyzed and separated. The results showed that the total extract was 650.20 g after acidification and alkalization, and then extracted with acidic chloroform and basic n-butanol, the three parts of extract were 3.01 g, respectively. The extraction rates of 0.53g ~ 23.13 g and the extraction rates of 1.51% ~ 0.27% ~ 11.57 g, respectively, indicating that the alkaloids were mainly concentrated in the extraction part of n-butanol, and mainly in the large polar alkaloids. TLC analysis showed that there were at least 5 alkaloids extracted from basic chloroform and alkaloids extracted from n-butanol. Compared with the standard sample, it was proved that the alkaloids contained in kumasu were mainly indole cetidine alkaloids such as coumarin.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.87

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