伯氏致病杆菌SN269菌株的筛选及次生代谢产物研究

发布时间：2018-06-16 22:16

  本文选题：伯氏致病杆菌 + 菌株分离　； 参考：《沈阳农业大学》2017年硕士论文

【摘要】：在昆虫病原线虫中寄生的共生菌属于肠杆菌科(Enterobacteriaceae)的革兰氏阴性细菌;其中与异小杆线虫科(Heterothabditidae)共生的发光杆菌(Phothrabdus),与斯氏线虫科(Steinernematidae)共生的致病杆菌(Xenohrabdus),是共生菌中仅有的的两类菌,他们寄生于3期侵染线虫的肠道内,随着寄主线虫一同进入到昆虫体内,并被释放到昆虫的血腔中,通过释放一系列的对昆虫有抑制分解作用的代谢产物,来杀死昆虫,为共生菌本身和线虫的生长发育和繁殖提供了充足的养分和养料。由于具有广泛的杀虫和抑菌效果,携带共生菌的线虫和线虫-共生菌复合体已经成为具有高效生物活性的新型的生物制剂,在农业上病虫害的生物防治中发挥着重要作用。特别是其中起到关键致病作用的共生菌,能够在发酵过程中合成和分泌多种具有高效抑菌活性和调节作用的代谢产物,对其进行分离纯化及结构鉴定在农业领域具有巨大的挖掘潜力,也将为新生物农药的研发奠定基础。本实验针对共生菌的主要研究方法和结果如下:1.共生菌菌株的分离纯化:本文通过对辽宁省内(抚顺市,丹东市凤城,铁岭市龙首山,铁岭市西丰等)以及省外周边(内蒙古牙克石市博克图,呼伦贝尔市扎兰屯;吉林省白山市长白山等)部分地区的土样进行采集,共得到102个土样;采用white-trap法分离获得了 38株初生型和4株次生型菌株,共计42株。2.目标菌株的筛选:对分离纯化得到的42株共生菌进行小发酵,用大孔树脂吸附法获取共生菌的粗提物。采用TLC检测技术对菌株发酵粗提物进行分析检测,结果表明SN231具有不同于其他所有粗提物的组分。分别采用菌丝生长速率法和菌块移置法测定候选菌株及其粗提物对植物病原真菌的抑制作用。结果表明,SN231菌株及其发酵粗提物均对辣椒疫霉病菌有一定的抑制作用。所以选定其为目标菌株,列入实验室的菌种库给予新的命名SN269,进行下一步实验。3.SN269菌株的鉴定:对SN269菌株进行16SrRNA序列分析。PCR扩增SN269菌株的16S rRNA序列,选择同源序列进行Clustal比对,采用Mega 6.0软件构建系统发育树,结果显示SN269菌株与Xenorhabdus bovienii聚在一起,同源性为100%,所以SN269 菌株属于 Xenorhabdus bovienii,命名为Xenorhabdus bovieniiSN269。4.SN269菌株次生代谢产物分离纯化:对SN269菌株进行液体发酵得到粗提物。然后利用柱层析技术与半制备高效液色谱技术相结合的方法对SN269菌株的次生代谢产物进行分离纯化,获得目标化合物D3。5.D3化合物的结构鉴定及活性研究:经核磁共振波谱及高分辨质谱分析,并结合相关文献,将D3鉴定为madumycin Ⅱ,是Streptogramin中具有高效抑菌活性的抗生素。通过菌丝生长速率法和微量肉汤稀释法测定化合物D3的抑真菌及细菌活性,结果表明化合物D3对辣椒疫霉和番茄灰霉的抑制效果尤为突出(EC50值分别为35.32和35.40μg/mL),并且对金黄色葡萄球菌具有很好的抑制效果IC50值为分别为0.13±0.02μg/mL。
[Abstract]:The symbiotic bacteria parasitized in the entomopathogenic nematodes belong to the Gram-negative bacteria of the Enterobacteriaceae (Enterobacteriaceae); among them, the symbiotic bacteria (Phothrabdus) symbiotic with the family nematonematonematonematonemataceae (Heterothabditidae) and the symbiotic Xenohrabdus (Xenohrabdus) symbiotic with the family of the family nematonemataceae (Steinernematidae) are the only two types of bacteria in the symbiotic bacteria. They parasitized in the intestinal tract of the 3 phase of the nematode infection. With the main line worms entered into the insect together and released into the insect's blood cavities, the insect was killed by releasing a series of metabolites that inhibited the insect's decomposition, which provided sufficient nutrients and nutrients for the growth and reproduction of the symbiotic bacteria itself and the nematode. The nematode and nematode symbiotic complex, which has a wide range of insecticidal and bacteriostasis, has become a new biological agent with high bioactivity and plays an important role in the biological control of agricultural pests and diseases. Secreting a variety of metabolites with high effective bacteriostasis and regulation, the separation and purification and structural identification have great potential in the field of agriculture, and will lay the foundation for the research and development of new biological pesticides. The main research methods and results for symbiotic bacteria in this experiment, such as the separation and purification of 1. symbiont strains, are adopted in this paper. The soil samples of Liaoning province (Fushun City, Dandong city Fengcheng, Tieling City, dragon head mountain, Tieling City Xifeng, Tieling City, Inner Mongolia, Yakeshi City, Hulun Buir city Zhalantun, Jilin province Baishan City Changbai Mountain) were collected and 102 soil samples were obtained. 38 primary and 4 strains were obtained by white-trap method. Secondary strains, a total of 42.2. target strains were screened: 42 strains of symbionts obtained by separation and purification were fermented and the crude extracts of the symbiotic bacteria were obtained by macroporous resin adsorption. The TLC detection technique was used to analyze the crude extracts of the strains. The results showed that SN231 was different from all the other crude extracts. The bacterial growth rate method and the bacterial block removal method were used to determine the inhibitory effect of the candidate strains and their crude extracts on the plant pathogenic fungi. The results showed that the SN231 strain and its fermented crude extract had certain inhibitory effects on the Phytophthora capsici. Step experiment.3.SN269 strain identification: 16SrRNA sequence analysis of SN269 strain,.PCR amplification of SN269 strain 16S rRNA sequence, select homologous sequence for Clustal comparison, and construct phylogenetic tree with Mega 6 software. The result shows that SN269 strain and Xenorhabdus bovienii are together and homology is 100%. Bdus bovienii, named as the secondary metabolite of Xenorhabdus bovieniiSN269.4.SN269 strain, was isolated and purified: the crude extract was obtained by liquid fermentation of the SN269 strain. Then, the secondary metabolites of the SN269 strain were separated and purified by the method of column chromatography and semi preparation high performance liquid chromatography, and the target compound D3.5. was obtained. The structure identification and activity study of D3 compounds: by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry, and combining related literature, D3 is identified as madumycin II, and it is an antibiotic with high bacteriostasis in Streptogramin. By mycelial growth rate method and micro broth dilution method, the antifungal and bacterial activity of compound D3 are determined, and the result table The inhibitory effect of D3 on Phytophthora capsici and gray mould was particularly prominent (EC50 value was 35.32 and 35.40 g/mL respectively), and the IC50 value of Staphylococcus aureus was 0.13 + 0.02 mu g/mL., respectively.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
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本文编号：2028289