本文選題：伯氏致病桿菌 + 菌株分離　； 參考：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：在昆蟲(chóng)病原線蟲(chóng)中寄生的共生菌屬于腸桿菌科(Enterobacteriaceae)的革蘭氏陰性細(xì)菌;其中與異小桿線蟲(chóng)科(Heterothabditidae)共生的發(fā)光桿菌(Phothrabdus),與斯氏線蟲(chóng)科(Steinernematidae)共生的致病桿菌(Xenohrabdus),是共生菌中僅有的的兩類菌,他們寄生于3期侵染線蟲(chóng)的腸道內(nèi),隨著寄主線蟲(chóng)一同進(jìn)入到昆蟲(chóng)體內(nèi),并被釋放到昆蟲(chóng)的血腔中,通過(guò)釋放一系列的對(duì)昆蟲(chóng)有抑制分解作用的代謝產(chǎn)物,來(lái)殺死昆蟲(chóng),為共生菌本身和線蟲(chóng)的生長(zhǎng)發(fā)育和繁殖提供了充足的養(yǎng)分和養(yǎng)料。由于具有廣泛的殺蟲(chóng)和抑菌效果,攜帶共生菌的線蟲(chóng)和線蟲(chóng)-共生菌復(fù)合體已經(jīng)成為具有高效生物活性的新型的生物制劑,在農(nóng)業(yè)上病蟲(chóng)害的生物防治中發(fā)揮著重要作用。特別是其中起到關(guān)鍵致病作用的共生菌,能夠在發(fā)酵過(guò)程中合成和分泌多種具有高效抑菌活性和調(diào)節(jié)作用的代謝產(chǎn)物,對(duì)其進(jìn)行分離純化及結(jié)構(gòu)鑒定在農(nóng)業(yè)領(lǐng)域具有巨大的挖掘潛力,也將為新生物農(nóng)藥的研發(fā)奠定基礎(chǔ)。本實(shí)驗(yàn)針對(duì)共生菌的主要研究方法和結(jié)果如下:1.共生菌菌株的分離純化:本文通過(guò)對(duì)遼寧省內(nèi)(撫順市,丹東市鳳城,鐵嶺市龍首山,鐵嶺市西豐等)以及省外周邊(內(nèi)蒙古牙克石市博克圖,呼倫貝爾市扎蘭屯;吉林省白山市長(zhǎng)白山等)部分地區(qū)的土樣進(jìn)行采集,共得到102個(gè)土樣;采用white-trap法分離獲得了 38株初生型和4株次生型菌株,共計(jì)42株。2.目標(biāo)菌株的篩選:對(duì)分離純化得到的42株共生菌進(jìn)行小發(fā)酵,用大孔樹(shù)脂吸附法獲取共生菌的粗提物。采用TLC檢測(cè)技術(shù)對(duì)菌株發(fā)酵粗提物進(jìn)行分析檢測(cè),結(jié)果表明SN231具有不同于其他所有粗提物的組分。分別采用菌絲生長(zhǎng)速率法和菌塊移置法測(cè)定候選菌株及其粗提物對(duì)植物病原真菌的抑制作用。結(jié)果表明,SN231菌株及其發(fā)酵粗提物均對(duì)辣椒疫霉病菌有一定的抑制作用。所以選定其為目標(biāo)菌株,列入實(shí)驗(yàn)室的菌種庫(kù)給予新的命名SN269,進(jìn)行下一步實(shí)驗(yàn)。3.SN269菌株的鑒定:對(duì)SN269菌株進(jìn)行16SrRNA序列分析。PCR擴(kuò)增SN269菌株的16S rRNA序列,選擇同源序列進(jìn)行Clustal比對(duì),采用Mega 6.0軟件構(gòu)建系統(tǒng)發(fā)育樹(shù),結(jié)果顯示SN269菌株與Xenorhabdus bovienii聚在一起,同源性為100%,所以SN269 菌株屬于 Xenorhabdus bovienii,命名為Xenorhabdus bovieniiSN269。4.SN269菌株次生代謝產(chǎn)物分離純化:對(duì)SN269菌株進(jìn)行液體發(fā)酵得到粗提物。然后利用柱層析技術(shù)與半制備高效液色譜技術(shù)相結(jié)合的方法對(duì)SN269菌株的次生代謝產(chǎn)物進(jìn)行分離純化,獲得目標(biāo)化合物D3。5.D3化合物的結(jié)構(gòu)鑒定及活性研究:經(jīng)核磁共振波譜及高分辨質(zhì)譜分析,并結(jié)合相關(guān)文獻(xiàn),將D3鑒定為madumycin Ⅱ,是Streptogramin中具有高效抑菌活性的抗生素。通過(guò)菌絲生長(zhǎng)速率法和微量肉湯稀釋法測(cè)定化合物D3的抑真菌及細(xì)菌活性,結(jié)果表明化合物D3對(duì)辣椒疫霉和番茄灰霉的抑制效果尤為突出(EC50值分別為35.32和35.40μg/mL),并且對(duì)金黃色葡萄球菌具有很好的抑制效果IC50值為分別為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.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
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本文編號(hào)：2028289