【作者】 钟凯；

【导师】 徐正君；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 放线菌在土壤中是微生物种群的重要组成部分,也是各种环境下容易分离得到的微生物。它能够生产出许多具有不同化学结构或生物活性的次生代谢产物。目前,已有很多由放线菌生产的物质被广泛应用于人类、动物疾病的治疗和农业上病虫害的防治上。近年来,由于很多放线菌类群被重复发现,预示着用新方法、新材料、新的活性物质筛选模型等进行放线菌分离是寻找新的次生代谢产物的途径之一。放线菌可以通过植物根际与植物形成寄生或共生关系。通过对特定植物的根际进行放线菌分离,是寻找新的次生代谢产物的手段之一。因此,本文以四川道地中药材姜黄根际为材料进行放线菌的分离,并利用2种活性物质筛选模型,对活性物质进行了初步研究,期望从姜黄根际中筛选到能生产具有生物活性的新次生代谢产物。1.利用水稻纹枯病菌和稻瘟病菌筛选拮抗活性放线菌本实验分离到了一株对水稻纹枯病菌和稻瘟病菌都有抑制活性的放线菌菌株,并命名为Eri1 2。通过形态学观察、生理生化指标的测定以及16S rDNA系统进化树分析,发现Eri1 2菌株属于灰色链霉菌(Streptomyces griseus)。该菌株的菌丝粗提物对水稻纹枯病菌和稻瘟病菌都具有很强的抑制活性。而且菌丝粗提物的田间小区试验发现,经喷施粗提物处理过的水稻植株的稻瘟病感病率明显下降,对稻瘟病叶病防治率可达64.5%。菌株Eri12产生的活性物质不溶于水,并具有良好的酸碱稳定性和热稳定性,经强酸、强碱和高温处理30min后,菌丝粗提物的抗菌活性基本不变。利用液相分配、固相萃取和高效液相色谱(HPLC)技术进行分离纯化,得到了3种活性物质。TOF-MS的分析测定表明,活性物质的分子量分别为219.1046,233.0837和390.2801。采用三重四级杆对其中对病原菌具有较强活性的物质(分子量为219.0416)进行了分析,通过分子片段比对及数据库分析,推测该活性物质中极有可能螯合锂元素,可能是新型抗生素。2.利用抗氧化试验筛选产抗氧化活性物质的放线菌利用抗氧化试验为筛选模型,从姜黄根际中分离到了一株产抗氧化剂的放线菌菌株,命名为Eri1 1.经形态学观察、生理生化指标测定以及系统进化树的构建,鉴定菌株Eri1 1属于链霉菌属。采用菲林试剂法和改良的氯化铝比色法对菌株发酵液中的总酚和总黄酮分别进行了测定,结果为每克菌株发酵液的干物质中含有相当于13.59±0.17 mg没食子酸的酚类物质和含有相当于9.93±0.83 mg芦丁的黄酮类物质。发酵液对ABTS自由基和OH自由基均有一定的清除能力。发酵液干物质对ABTS自由基和羟自由基的清除率为50%时的浓度分别为223.81士24.50μg/mL (IC50=223.81士24.50μ/mL)和582.42士83.10μg/mL(IC50=582.42士83.10μg/mL).通过对菌株发酵液抗氧化活性的初探,说明菌株Eri1 1具有生产抗氧化剂的能力。通过不同的筛选模型,我们从姜黄根际中分离出了2株产不同生物活性物质的放线菌菌株。本文中发现其中一株能代谢产生一种可能含有锂元素的活性物质,对水稻纹枯病菌和稻瘟病菌有较强的抑制效果,推测其是一种新型的抗生素。在今后的工作中,我们将对该活性物质的结构进行鉴定分析,以及研究它对水稻稻瘟病菌和水稻纹枯病菌的作用机理。更多还原

【Abstract】 Actinomycetes are widely distributed in a variety of natural and manmade environments, constituting a very important component of the microbial population in most soils. Actinomycetes play a significant role in the pharmaceutical industry for their capacity to produce secondary metabolites with the diverse biological activities and chemical structures. Tens of thousands of such compounds have been isolated, purified and characterized, many of which have been developed into drugs for treatment of wide range of diseases in human, veterinary and agriculture sectors. Recently, the rate of discovery of new actinomycete has decreased, whereas the rate of re-isolation of known material has increased. Therefore, many efforts have been made to select and isolate the new actinomycetes from other biotopes.Actinomycetes are known to form intimate associations with plants and colonize their internal tissue, and they inhabit a wide range of plants as either symbionts or parasites from the plant rhizosphere. Isolating the actinomycetes from the rhizosphere of the special plants is the one kind of methods for searching for the novel second metabolites with biological activity. Therefore, we focused on isolating the actinomycetes from the rhizosphere of Rhizoma Curcumae Longae, one kind of typical Chinese medical plants in Sichuan province, by using two kinds of different screening system. The main results are provided as follows:1. Isolation actinomycetes by using antibacterial systemIn the present research, we isolated one kinds of actinomycetes strain which have strong antagonism activity against rice sheath blight(Rhizoctonia solani) and rice b\ast(Magnaporthe grisea), and named as Eri12. According to the morphology, physiological and chemical tests and phylogenetic tree based on the 16S rDNA, the strain Eri12 was characterized as Streptomyces griseus.The mycelia extract with 80% methanol also showed strong antagonism activity against rice sheath blight(Rhizoctonia solani) and rice blast(Magnaporthe grisea). The field test was adopted with the mycelia extract for testing the ability of preventing rice from rice blast(Magnaporthe grisea). The blast susceptibility rate of the rice plants sprayed with the mycelia extract decreased significantly, and the rice plants infectious rate of rice blast (Magnaporthe grisea) was 35.5% after treated with mycelia extract.The active compounds produced by the strain Eri12 is insoluble in the water. The active compounds were stable after treated with acid, alkali and 100℃, respectively. Purification of the active compounds was achieved by using extraction, solid partition extract and HPLC. After purification, there were three active compouds. The molecular weight of active compounds were 219.1046,233.0837 and 390.2801 separately, by using the LC-TOF/MS. And the compound (MW: 219.1046) showed strongest activity was submitted to Quadrupole. As a result, there could be Li element in the compound (MW: 219.1046). Therefore, the active compound (MW: 219.1046) could be a novel candidate of the antibiotics with Li element.2. Isolation of actinomycetes based on the antioxidant activityThe one of actinomycete strains was isolated from the rhizosphere of-Rhizoma Curcumae Longae by using antioxidant assay system, and named as Erill. According to the morphology, physiological and chemical tests and phylogenetic tree based on the 16S rDNA, the strain Erill was strongly characterized as Streptomyces.The total contents of phenols and flavonoids of cultured broth were investigated by using the Folin-Ciocalteu method and AICl3 colormetric method, respectively. The total phenols contents in the cultivated broth were calculated to be 13.59±0.17 mg gallic acid equivalent/g extract. Meanwhile, by using AICI3 colormetric method, the total flavonoids contents in the cultivated broth were calculated to be 9.93±0.83 mg rutin equivalent/g extract.The cultured broth had radical scavenging activity with both ABTS radicals and Hydroxyl radicals. The half-inhibitory concentration (IC50) of ABTS and hydroxyl radicals scavenging activity were estimated at 223.81±24.50μg/mL and 582.42±83.10μg/mL, respectively.As a result, the strain Erill could metabolize some antioxidants, which suggested the strain Eril 1 could be the potential resource for the antioxidant industry.Using different screening system, we isolated two Streptomyces strains metabolizing different active compounds from the rhizosphere of Rhizoma Curcumae Longae. Most importantly, we purified one of the compound with strong antagonism activity against rice sheath blight (Rhizoctonia solani) and rice blast (Magnaporthe grisea). And after analyzed with instruments, the compounds is assumed to be a novel antibiotic with Li element. Because of the novelty of the compound, the thorough studies of structure identification and the mechanism of the antagonism activity against the pathogens will carry on.更多还原