本文選題：南極菌Psychrobacter + sp.B-3�。� 參考：《青島科技大學》2011年碩士論文

【摘要】：南極因其干燥、酷寒、強輻射等自然環(huán)境,生存于此的微生物有獨特的分子生物學機制和生理生化特征,能產(chǎn)生新型的活性物質(zhì),如胞外多糖。胞外多糖在南極海冰中可以保護其生物體免受冰晶傷害,降低其它化學物質(zhì)的傷害。南極微生物胞外多糖在南極生態(tài)系統(tǒng)中具有重要作用,但其潛在的生物生化活性物質(zhì)應(yīng)用研究報道還很少。本實驗以中國第24次南極科學考察烏拉圭站(S62°11’50.52”W58°55’50.4")采集的海冰樣品,經(jīng)佐貝爾2216E培養(yǎng)基篩選,分離獲得一株產(chǎn)高糖南極菌B-3,經(jīng)16SRNA鑒定為嗜冷菌Psychrobacter,通過對該菌的培養(yǎng)條件優(yōu)化,胞外多糖分離純化獲得高純度樣品；采用高效凝膠滲透色譜(HP-GPC)分析確定B-3胞外多糖的組成；對B-3多糖的植物誘導活性進行研究,以期為南極菌的應(yīng)用開發(fā)提供科學依據(jù)。 1.對57株南極菌的外形觀察、剛果紅法和顯微鏡檢測法篩選出一株產(chǎn)糖菌。16S rDNA鑒定結(jié)果表明,南極海洋菌B-3是一株嗜冷菌Psychrobacter sp.B-3。由于嗜冷菌是南極生態(tài)系統(tǒng)中常見的一類細菌,所以對其進行進一步的研究。 2.南極嗜冷菌B-3最佳的實驗室產(chǎn)糖條件是：最佳碳源,1%的葡萄糖；最佳氮源,0.5%的黃豆粉；最佳NaCl濃度,3%；最佳溫度,10℃；最佳pH值為7；培養(yǎng)時間,60 h。試驗結(jié)果表明,南極海洋菌在4-25℃下均能正常生長,因此確定該菌為嗜冷菌,即南極嗜冷菌B-3。100L發(fā)酵罐結(jié)果表明,其條件與實驗室培養(yǎng)條件大體相同,其胞外多糖產(chǎn)量比實驗室發(fā)酵有所提高。 3.南極嗜冷菌B-3發(fā)酵液經(jīng)過95%乙醇沉淀、sevege法去蛋白后獲得粗多糖。獲得的B-3多糖粗品經(jīng)Sephadex G-50凝膠層析進一步純化,獲得純品。以高效凝膠滲透色譜(HP-GPC)測定多糖分子量分別為：2112Da。水解后純品的糖氰乙酸酯衍生物經(jīng)氣相色譜分析,確定其單糖組成為甘露糖和葡萄糖。 4.該實驗用0.3%、0.5%和0.8%(或0.2%、0.4%、0.8%、1.()%)的B-3胞外寡聚糖對黃瓜幼苗分別噴施不同時間后,黃瓜體內(nèi)的幾丁質(zhì)酶,β-1,3-葡聚糖酶,苯丙氨酸解氨酶(PAL),過氧化物酶(POD)、過氧化氫酶(CAT)、谷胱甘肽-S轉(zhuǎn)移酶(GSH-ST)、谷胱甘肽過氧化物酶(GSH-PX)等活性均較對照有不同程度的提高,并且與0.5%的殼聚糖具有相似的誘導效果；同時,0.5%的B-3多糖能夠顯著的降低黃瓜白粉病的病情指數(shù),防效達24.49%,B-3寡聚糖有望開發(fā)為新型抗病誘導子。 5對南極嗜冷菌B-3胞外多糖的低溫保護作用的測定表明,不同的濃度南極嗜冷菌B-3胞外多糖,均可以降低溶液的冰點。研究還表明,重復(fù)的凍融循環(huán)后,胞外多糖保護菌體免受對其造成的傷害,使該菌保持較好的生長能力,所以推測該胞外多糖具有一定的低溫保護作用。 綜上所述,南極嗜冷菌B-3的胞外多糖經(jīng)氣相色譜分析是由葡萄糖和甘露糖組成；該糖能夠黃瓜幼苗的抗病性,降低黃瓜白粉病的發(fā)病率；同時,該菌最佳產(chǎn)糖溫度為10℃、鹽度為3%,其產(chǎn)生的胞外多糖還能夠降低冰點,避免重復(fù)凍融對菌體的傷害作用。本結(jié)果為研究南極微生物的應(yīng)用生產(chǎn)提供了依據(jù),以及南極資源的開發(fā)利用提供了科學依據(jù)。
[Abstract]:Antarctica, because of its natural environment, such as dry, cold and strong radiation, has a unique molecular biological mechanism and physiological and biochemical characteristics, which can produce new active substances, such as extracellular polysaccharide. Extracellular polysaccharide can protect its organisms from ice crystals in the Antarctic sea ice and reduce the damage of other chemical substances. Antarctic microbes Extracellular polysaccharide (exo) plays an important role in the Antarctic ecosystem, but the potential application of biobioactive substances is rarely reported. In this experiment, the sea ice samples collected from the twenty-fourth Chinese Antarctic scientific survey Uruguay station (S62 11 '50.52 "W58 55' 50.4") were selected by the screening of the Zoar 2216E medium, and a high sugar South was obtained. B-3 was identified as eosinophilic bacteria Psychrobacter by 16SRNA, and high purity samples were obtained by optimizing the culture conditions of the bacteria and separation and purification of extracellular polysaccharide. The composition of B-3 extracellular polysaccharide was determined by high performance gel permeation chromatography (HP-GPC) analysis, and the plant induced viability of B-3 polysaccharide was studied in order to provide the application development of Antarctic bacteria. Scientific basis.
1. to observe the shape of 57 strains of Antarctic bacteria, the results of.16S rDNA identification of a strain of sugar producing bacteria by Congo red method and microscope detection show that the Antarctic marine bacteria B-3 is a cold strain Psychrobacter sp.B-3., which is a common type of bacteria in the Antarctic ecosystem because of the eosinophilia.
2. the best laboratory sugar producing conditions for Antarctic eosinophilic bacteria B-3 are: the best carbon source, 1% glucose, the best nitrogen source, 0.5% soybean powder, the best NaCl concentration, 3%, the optimum temperature, 10 centigrade, and the optimum pH value of 7; the incubation time, the 60 h. test results show that the Antarctic marine bacteria can grow normally at 4-25 C, thus determining that the bacterium is eophilia, that is, The results showed that the conditions of the Antarctic cold B-3.100L bacteria fermentation tank were similar to those of the laboratory culture conditions, and the yield of extracellular polysaccharides was higher than that of laboratory fermentation.
3. the fermentation broth of Antarctic eosinophilic bacteria B-3 was precipitated through 95% ethanol, and crude polysaccharides were obtained by sevege method. The purified B-3 polysaccharide was purified by Sephadex G-50 gel chromatography and obtained pure products. The molecular weight of polysaccharide was determined by high performance gel permeation chromatography (HP-GPC). The monosaccharide composition was identified as mannose and glucose by spectral analysis.
4. in this experiment, the chitinase, beta -1,3- glucanase, phenylalanine ammonia lyase (PAL), peroxidase (POD), peroxidase (CAT), glutathione -S transferase (GSH-ST), glutathione peroxidase (GSH), glutathione peroxidase (GSH), and glutathione peroxidase (GSH) were applied to cucumber seedlings with 0.3%, 0.5% and 0.8% (or 0.2%, 0.2%, 0.4%, 0.8%, 1. ()%). -PX) and other activities were improved in varying degrees and similar to 0.5% of chitosan. At the same time, 0.5% B-3 polysaccharide could significantly reduce the disease index of cucumber powdery mildew, and the control effect was 24.49%. The B-3 oligosaccharide was expected to be a new type of disease resistant inducer.
5 the determination of the low temperature protection of the Antarctic eosinophilic B-3 extracellular polysaccharide showed that the different concentrations of the Antarctic eosinophilic B-3 extracellular polysaccharide could reduce the freezing point of the solution. The study also showed that after the repeated freezing and thawing cycle, the extracellular polysaccharide protected the mycelium from the damage caused by the bacteria, so that the bacteria maintained good growth ability, so that the extracellular polysaccharide could be maintained. Polysaccharide has a certain protective effect on low temperature.
To sum up, the GC analysis of the extracellular polysaccharide of Antarctic cold strain B-3 is composed of glucose and mannose, which can resist the disease of cucumber seedlings and reduce the incidence of cucumber powdery mildew. At the same time, the optimum temperature is 10 C and the salinity is 3%. The production of extracellular polysaccharide can also reduce freezing point and avoid repeated freezing and thawing bacteria. This result provides a scientific basis for the study of the application and production of Antarctic microorganism and the exploitation and utilization of Antarctic resources.

【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R378

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