【作者】 祝凯；

【导师】 王一丁；

【作者基本信息】 四川师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 本文在对四川彭州产华重楼的内生菌进行全面分离筛选的基础上，通过体外拮抗试验筛选出一株具有广谱抗菌特性的的内生菌菌株，对其进行了菌种的分子鉴定，命名为(Paenibacillus daejeonensis)SS02。通过采用不同的培养温度、培养时间、pH值对华重楼内生细菌SS02进行培养条件优化，对其抗菌活性进行检测，得到了最佳发酵条件为：在pH值为7.0的PB培养基中30℃、摇床振荡培养72小时后，用饱和度20-50％为硫酸铵对其进行分级盐析，可得最高发酵活性物质。将发酵液经12000rpm离心10分钟，去除残渣及菌体。培养液经硫酸铵分级盐析后，发酵原液活性丧失，同时在盐析沉淀物中检测到高抗菌活性，离心后收集沉淀，将沉淀溶解于磷酸缓冲液中，获得粗提蛋白。粗提蛋白经过Sephadex G-75分子筛凝胶柱层析和DEAE-32纤维素柱层析脱盐后，分离收集不同的蛋白组分，并以植物病害菌作跟踪试验比对，分离纯化出一种抗菌蛋白，用SDS-PAGE测定提取物纯度表明获得蛋白质纯品，进行分子量测定，得到电泳结果为单一条带，此蛋白分子量为56000，聚丙烯酰胺凝胶等电聚集电泳(PAGE-IEF)等电点为6.4，对热和紫外线稳定，对蛋白酶部分敏感。纯化后的SD22对玉米纹枯菌(Rhizoctonia cerealis)、油菜菌核病(Sclero-tinia sclerotiorum)、苹果轮纹病菌(Physalospora piricala)、绿色木霉(Trich-odema viride)、绿色粘帚霉(Gliocladium viride)、弯孢霉菌(Curvularia leaf-spot)、镰刀霉菌(Fusarium sp.)、赤霉菌(Fusarium head blight)、球孢白僵菌(Beauveria bassiana)、大肠杆菌(Escherichia coli)、金黄色葡萄球菌(St-aphylo-coccus aureus)、枯草芽孢杆菌(Bacillus subtilis)、白色念珠菌(Cand-idal vaginitis)、冬瓜枯萎菌(Fusarium oxysporum Schl.emend.Snyder &Han-se)等菌有很强的抑制作用。更多还原

【Abstract】 In this thesis, a broad-spectrum antibiotic Endophytic strain was screened outby antifungal exogenous experiments, basing on seperating Endophytic BacteriaSS02 of Paris Polyphylla Var. Chinensis comprehensively, then it was named SS02after molecular identifying.SS02 was incubated through different temperature, time and PH, and theantibacterial competence was checked, then an conclusion was come to: the bestcondition of fermenting the protein is incubating it in PB at 30℃, 7.0PH, shaking it72 hours in bassinet and purifying it throughing on 20%-50%（NH₄）₂SO₄.The zymolytic liquor was putted in centrifuge, 10 min lasted and12000rpm, and the residua and thalli was cleared. After purified throughing on（NH₄）₂SO₄, the antibacterial competence of zymolytic liquor was gone, andsynchronously, the obvious antibacterial competence was detected indeposit. When the deposit was gathered after centrifuge and it was dissolved inphosphate buffer, the original protein was gained. Then a antibacterial protein wasseperated when different protein was gathered through purifying original protein ingel pole and fibre pole respectively and contrasted against plant pathogen by trackexperiment. The SDS-PAGE shown that the protein is pure. Its molwt wasmensurated—56000 and a single stripe was shown in electrophoresis. Its PI valueis 6.4 in PAGE-IEF, it is thermostabale, stable in ultraviolet and partially sensitiveto enzyme. Purified SD22 can strongly restrain strains such as Rhizoctonia cerealis,Sclerotinia sclerotiorum Physalospora piricala, Trichodema viride, Gliocladiumviride, Curvularia leaf spot, Fusarium sp, Fusarium head blight, BeauveriaBassiana, Escherichia coli, Staphylo-coccus aureus, Bacillus subtilis, Candidalvaginitis, Fusarium oxysporum Schl.emend.Snyder&Hansem.更多还原