论文标题:砂地柏内生真菌初步研究
Isolation of Endophytic Fungi from Medicinal Plant Sabina Vulgaris Ant. and Preliminary Studies on Metabolites
论文作者
论文导师 张兴,论文学位 硕士,论文专业 农药学
论文单位 西北农林科技大学,点击次数 70,论文页数 59页File Size2391K
2006-06-01论文网 http://www.lw23.com/lunwen_1002278832/
Sabina vulgaris Ant.; Endophytic fungi; Podophyllotoxin analogues; Bioachitiies Alternaria
砂地柏Sabina vulgaris Ant为柏科Cupressaceae圆柏属Sabina常绿葡匐灌木,是一种重要的药用和杀虫植物。内生真菌作为一类特殊的微生物类群,能够产生许多结构新颖、生理活性独特的或与宿主植物相同和相似的天然生理活性物质的特点,因而在天然药物领域具有巨大的开发潜力。基于此,本研究对砂地柏内生真菌进行了初步研究。主要结果如下: (1)采用常规分离方法,在4种培养基上,对砂地柏不同年份茎、叶和果实,根和茎的木质部和韧皮部等组织的内生真菌进行了分离纯化,从中分离得到126株内生真菌。结果显示砂地柏内生真菌菌株分布随树龄的增长而增加;在茎和叶组织中菌株分布多于其它组织。PDA和CA培养基比其他培养基更利于砂地柏内生真菌的分离。 (2)采用小叶碟添加法和浸毒叶片法,对分离得到的内生真菌杀虫活性筛选。结果表明,在菌株代谢产物浓度为20mg/mL的剂量下,有28株内生真菌发酵产物对三龄初期粘虫表现出拒食或毒杀活性,其中14株拒食率或死亡率大于50%;有24株内生真菌对三龄初期小菜蛾表现出拒食或毒杀活性,其中7株拒食率或死亡率大于50%。 (3)以砂地柏代表性次生代谢产物苦鬼臼毒素、鬼臼毒素和脱氧鬼臼毒素为外标物,对菌株发酵产物进行HPLC检测分析,结果显示,从6株菌株的代谢产物中发现可能含有鬼臼类物质或与鬼臼类物质具有相似UV特征的化合物。 (4)杀虫活性筛选结果发现菌株LP1-3对三龄初期小菜蛾表现出现对较高的毒杀活性;同时HPLC检测分析的结果显示菌株LP1-3代谢产物中可能含有与鬼臼类物质具有相似UV特征的化合物;而且菌株LP1-3的发酵产量相对较高,约232.5mg/L。因此确定对菌株LP1-3进行进一步研究。 (5)对菌株LP1-3的代谢产物进行初步分离纯化,得到一个纯品化合物。通过对该化合物质谱、红外光谱、紫外光谱、核磁共振谱的初步分析,推测该化合物可能为一种木脂素类化合物。采用小叶碟添加法测定了该化合物对三龄初期粘虫的拒食活性,其48h拒食中浓为959.7mg/L,略低于鬼臼毒素和脱氧鬼臼毒素的活性。 (6)结合形态观察结果,对菌株LP1-3的ITS-5.8S rDNA区域核苷序列在GenBank中的进行比对,该菌株同7株链格孢属真菌的同源性达到99~100%,根据系统发育树和遗传距离分析,初步鉴定菌株LP1-3为链格孢属真菌。
The evergreen prostrate shrub Sabina vulgaris Ant. (syn.: Juniperus sabina Linnaeus) belonging to the genus Sabina ( family Cupressaceae) is a sort of traditional chinese herbs , which is widely distributed in the northwest of China. This studies isolated of endophytic fungi from plant Sabina vulgaris Ant. and studies on strains’metabolites. The culture were extracted with chloroform, the bioassay activity of the metabolites assayed with leaf-disk method and Leaf Dipped Method, and analysed by HPLC. The results were described as following: (1) Used the general methods. 126 strain endophytic fungi isolated from healthy organism of pharmaceutical plant Sabina vulgaris Ant. The results of the Isolation shown that the distributing was more in the veteran,and more in the stem & leafl. Culture medium include PDA and CA are becoming to isolate the endophytic fungi. (2) The concentration of the extraction from strain’s fermentation were 20mg/mL. The bioassay activity against the 3rd larvae of Mythimna separate Walker. and Plutella xylostella L.were assayed with leaf-disk method and Leaf-Dipped method. The results shown that there are 28 strains had the bioassay activity against the 3rd larvae of Mythimna separate Walker., and 24 strains had the bioassay activity against the 3rd larvae of Plutella xylostella L. (3) The metabolites were analysed by HPLC. PicroPodophyllotoxin, Podophyllotoxin and Deoxypodophyllotoxn were three Standard Samples with HPLC. The result shown that there were 6 strains had the Similar tR and Characteristics of UV with the Picropodophyllotoxin, Podophyllotoxin and Deoxypodophyllotoxn. (4) The results of bioassay activity shown that the metabolites of strain LP1-3 had poison activity against 3rd larvae of Plutella xylostella L. and results HPLC shown that metabolites of strain LP1-3 had the more values tR and Characteristics of UV with the Standard Samples. Based on the bioassay activity and results with HPLC, strain LP1-3 was selected to be the continuation. (5) By isolation technique of Natural Products. One compound was obtained from the metabolites of strain LP1-3. The NO.compd A had some activities the AFC50 against the 3rd
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