苦馬豆內(nèi)生真菌分離鑒定及其生物堿成分分析
發(fā)布時間：2018-11-07 07:43
【摘要】：1.苦馬豆內(nèi)生真菌分離與鑒定為探明苦馬豆中是否存在內(nèi)生真菌及其內(nèi)生真菌菌落與分布情況,本研究對采自內(nèi)蒙古阿拉善盟草原的苦馬豆樣品采用組織分離法對內(nèi)生真菌進行分離,運用傳統(tǒng)形態(tài)學(xué)與分子生物學(xué)方法對所得菌株進行種屬鑒定。此外,為了獲得較好的分離效果,本次試驗也對苦馬豆不同組織的最佳消毒時間進行確定,發(fā)現(xiàn)苦馬豆莖和種子用75%乙醇及2%NaClO最佳表面消毒時間分別為90s、5min,40s、2.5min。本次試驗從苦馬豆的莖、葉、葉柄和種子進行分離共得到70株內(nèi)生真菌,其分屬于交鏈孢屬(Alternaria Nees)、鐮刀孢屬(Fusarium sp.)、枝孢屬(Cladosporium sp.)、暗梗單孢霉屬(Chloridium Lk.)、殼色單隔孢屬(Diplodia sp.)、隔孢腔菌屬(Pleosporales sp.)、小雙胞腔菌(Didymella fabae)、Laetisaria arvalis和Preussia australiis等9個屬。并且有3株鑒定到種為Didymella fabae、Laetisaria arvalis和Preussia australiis。Alternaria Nees的相對分離頻率最高(61.43%),為苦馬豆內(nèi)生真菌的優(yōu)勢菌屬。各樣品組織中,葉的分離率(19.58%)最高,種子(41.39%)次之,表明種子和葉是內(nèi)生真菌侵染和定殖的主要部位。2.苦馬豆化學(xué)成分系統(tǒng)預(yù)試驗按照植物化學(xué)成分系統(tǒng)預(yù)試的常規(guī)方法,對苦馬豆的化學(xué)成分進行了系統(tǒng)預(yù)試驗。結(jié)果表明:苦馬豆含有甾體、萜類、揮發(fā)油、油脂、糖、有機酸、氨基酸、肽、蛋白質(zhì)、生物堿、黃酮、香豆素、內(nèi)酯類化合物、皂苷、鞣質(zhì)、酚類、強心苷類、蒽醌類。3.苦馬豆生物堿TLC分析本試驗采用TLC分析、平面色譜圖像定量分析軟件及柱層析分離等技術(shù),對苦馬豆生物堿成分進行分析和分離。結(jié)果表明,苦馬豆5.00 kg乙醇熱回流提取得總浸膏650.20 g,經(jīng)酸化、堿化,所得堿液依次用酸性氯仿、堿性氯仿、堿性正丁醇萃取,得到三部分浸膏分別為3.01g、0.53g、23.13 g,提取率分別為1.51%、0.27%、11.57%,說明苦馬豆生物堿主要集中在正丁醇提取部分,以大極性生物堿為主。對各部分提取物進行薄層層析分析,結(jié)果顯示堿性氯仿和堿性正丁醇提取部分生物堿至少分別有5種。經(jīng)與標(biāo)準(zhǔn)樣品對照,證明苦馬豆所含的生物堿主要以苦馬豆素等吲哚里西啶生物堿。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.87
本文編號：2315674
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.87
【共引文獻】
相關(guān)期刊論文 前3條
1 王帥;張玲;陳根元;席琳喬;馬春暉;;小花棘豆生態(tài)學(xué)研究概況[J];家畜生態(tài)學(xué)報;2014年03期
2 張曉晶;劉偉霞;魏鵬;格桑羅布;確生;張梁;張慶英;;藏藥鐮形棘豆毒性及化學(xué)成分相關(guān)性分析[J];中國中藥雜志;2014年07期
3 周啟武;于龍鳳;路浩;曹丹丹;趙寶玉;;小花棘豆和變異黃芪內(nèi)生真菌顯微分布及定量檢測[J];微生物學(xué)報;2014年05期
相關(guān)碩士學(xué)位論文 前3條
1 劉鵬;甘肅棘豆內(nèi)生細菌中苦馬豆素降解菌的篩選與鑒定[D];四川農(nóng)業(yè)大學(xué);2011年
2 陳基萍;瘋草內(nèi)生真菌種屬鑒定及次生代謝產(chǎn)物研究[D];西北農(nóng)林科技大學(xué);2012年
3 鄧?yán)?苦馬豆素降解菌的篩選與鑒定[D];塔里木大學(xué);2015年
，本文編號：2315674
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