【作者】 张宝；

【导师】 李平兰； 尚庆茂；

【作者基本信息】 中国农业大学 ， 食品生物技术， 2014， 博士

【摘要】 本研究以从柠檬样品中筛选分离到一株能够抗立枯丝核菌的解淀粉芽孢杆菌K103为试材,对其所产脂肽的分子结构、生化特性、抗菌机理及植物生防作用进行深入研究,为该抗菌脂肽的生产与运用提供理论基础与技术支持。所得主要研究结果与结论如下：(1)通过形态学鉴定、生理生化实验及16S rDNA的分子生物学等方法,对产抗菌脂肽的K103菌株进行了分类学鉴定,最终确定菌株K103为解淀粉芽孢杆菌,并将其命名为解淀粉芽孢杆菌K103(Bacillus amyloliquefaciens K103).(2)解淀粉芽孢杆菌K103发酵上清液经酸沉醇提所得脂肽粗提物,经SephadexLH-20凝胶层析、制备型C18反相高效液相层析分离纯化后,得到两种具有强烈抑制立枯丝核菌的纯化组分P3、P4。采用氨基酸组成分析结合串联质谱的方法对抗菌纯化组分P3、P4的结构进行分析鉴定,最终确定组分P3、P4分别为含有14个和15个碳原子脂肪酸链的bacillomycin L类脂肽同系物。(3)特性分析结果显示,bacillomycin L抗菌谱广,稳定性高,对热、酶不敏感,能抗紫外照射,在食品防腐和植物生物防治方面有巨大的应用潜力。(4)荧光染料实验、电镜实验、人工模拟膜实验结果显示,脂肽bacillomycin L处理立枯丝核菌菌丝体细胞后,细胞表面粗糙,细胞内部原生质团聚、分布不均匀以及胞内物质流失,细胞形态改变、结构破坏,细胞膜通透性和完整性破坏,膜流动性增加。(5)凝胶阻滞实验和光谱吸收滴定实验表明,bacillomycin L可与立枯丝核菌DNA在体外非特异性结合,说明bacillomycin L可能作用于胞内其它目标物质。(6)SDS-PAGE电泳分析发现bacillomycin L处理前后各分子量范围未发生条带缺失,但条带变浅；Native-PAGE电泳分析发现,bacillomycinL处理非变性可溶性蛋白条带差异较为明显,出现部分条带缺失、变浅情况。(7)以未处理立枯丝核菌菌丝体胞内总蛋白2-DE图谱为参考图谱,bacillomycinL处理后,差异倍数大于2倍且有显著性差异的蛋白点共有48个,明显上调的点有43个,明显下调的蛋白点有5个。对这些差异蛋白质点进行了质谱鉴定及功能分析表明,这些蛋白质主要参与糖代谢,核苷酸代谢,氨基酸代谢,跨膜运输,抗逆反应相关蛋白,转录,核苷酸结合和蛋白降解以适应胁迫条件。部分差异表达蛋白的qRT-PCR分析和ATPase活性分析实验,蛋白所对应基因表达的变化和酶活性的变化,与相应差异蛋白质表达的变化趋势基本一致。(8)通过盆栽试验,研究根际接种bacillomycinL和芽孢杆菌K103对黄瓜幼苗生长、养分吸收利用和立枯病防治的效应研究。施用bacillomycin L(500μg/ml)和芽孢杆菌K103能显著促进黄瓜苗期植株生长,提高植株养分吸收,有效防控立枯病发生。更多还原

【Abstract】 In order to provided technical support and theoretical basis for the development and application of lipopeptide antibiotics as a biological control agent, the purification, identification, physical and chemical characteristics, mechanism of action along with application in biocontrol of bacillomycin L produced by Bacillus amyloliquefaciens K103, isolated from a lemon sample and suppressing Rhizoctonia solani Kiihn, were investigated in detail.(1)The organism of interest was isolated from the surface of a lemon sample, identified as a B. amyloliquefaciens strain, and designated as B.amyloliquefaciens K103using biochemical, physiological, and16S ribosomal DNA sequencing.(2)Two antifungal compounds were purified from the culture broth using acid precipitation, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Based on its amino acid composition and MS/MS spectrometry analysis, they were successfully identified as bacillomycin L and its derivatives, varying only in terms of the length of the fatty acid aliphatic chains from C14to C15.(3)Results obtained from charateristics analysis of bacillomycin L shows it has a broad spectrum, high stability to heat, enzyme, and ultra-vioket radiation,which indicated it has a potentail application in biological control and food conservation.(4)Various fluorescent techniques, electron microscopy and model membrane assay show that the surface of cells was rough with more wrinkles and vesicles, the membranes show deformation and materials within the cytoplasm seem to be shrunken an unevenly dispersed, the fluidity of membrane was increased and the integrity and permeabilization of membrane was disrupted.(5)Gel retardation experiments and absorption titrations assay showed that bacillomycin L could bind to R. solani hyphal cells DNA in vitro,indicating that bacillomycin L was likely to interaction with the intracellular targets.(6) The protein bands of R. solani hyphal cells detected by SDS-PAGE were fleeted but not disappeared. Native-PAGE showed that non-denaturation protein bands of R. solani hyphal cells were diffenrent markedly with some bands fleeted and disappeared.(7)As compared with the untreated sample, there were48spots whose abundence varied significantly (more than2-fold),43spots significantly increased and5spots decreased after treated by bacillomycin L. matrixassisted laser desorption/ionization time-of-flight tandem mass spectrometry(MALDI-TOF/TOF-MS) and function classification analysis indicated these protein were involved in carbohydrate metabolism, nucleotide metabolism, amino acids metabolism, transmembrane transport, stress response,transcription, nucleotide binding, protein degradation, and cellular component organization. Real-time PCR and ATPase activity analysis of the hyphal proteins differentially expressed indicated that these differentially expression were consistented with corresponding gene expression roughly. (8)A pot experiment was carried out to investigate the effects of bacillomycin L and Bacillus amyloliquefaciens K103on the growth of cucumber seedlings, absorption and utilization of nutrient and the suppression of the seedlings damping-off disease. The results indicated that the application of bacillomycin L and Bacillus amyloliquefaciens K103could effectively promote growth, nutrition absorption of banana seedlings, and reduce the occurrence of seedlings damping-off.更多还原