多粘类芽孢杆菌KF-1抗真菌物质的纯化及其编码基因簇分析

发布时间：2018-01-11 15:42

本文关键词：多粘类芽孢杆菌KF-1抗真菌物质的纯化及其编码基因簇分析　出处：《济南大学》2016年硕士论文　论文类型：学位论文

【摘要】：菌株KF-1由本实验室分离,通过形态观察、生理生化特征以及16S rDNA序列分析将KF-1鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa),抑菌实验显示KF-1对多种病原真菌具有很好的抑菌活性。产抑菌活性物质培养优化实验结果显示PDA培养基为最佳选择。进一步采用乙酸乙酯法萃取菌株KF-1胞外抑菌活性物质,电喷雾质谱法(ESI-MS)分析显示以上萃取物中含4组具有抗菌活性物质的信号峰,分别为:峰1保留时间为14.753 min,分子量为188,可能为抑菌化合物3,5-二羟基苯甲酸甲酯[65]的分子量一致;峰2保留时间为22.676min,产量非常高,并且对革兰氏阳性菌以及真菌均有着很好的抑制效果,其离子峰[M+Na]+及二倍离子峰[2M+Na]+质荷比分别为413.8、803.6,从而推断该分子量为390,属于大环内酯类小分子抗生素;峰3主要包括两组化合物,分子量分别为251和453,通过与已知抗生素分子量比较,与核苷类抗生素Polyoxin的分子量相类似,其抑菌结果显示该化合物可抑制白色念珠菌的生长,属于抗真菌类抗生素。峰4主要包括分子量为882.4以及933.5的化合物,其分子量为882与LI-F脂肽家族中的Fusaridins A[M+H]+相一致,而933分子量则与LI-07F化合物类似。接下来利用全基因组鸟枪法,对多粘类芽孢杆菌KF-1的全基因组序列进行了测序分析。结果显示:KF-1基因组大小为5.1 Mb,GC含量46.26%,共编码5229个ORF,其中ORF总长度占基因组长度的84.53%,另外有6组16S/5S/23S rRNA操纵子区域。同时鉴定了107个非编码tRNA,其序列总长度为8275 bp,占基因组总长度的0.001429792%。基因组测序结果已提交到Gen Bank,登录号为LNZF00000000。通过GO注释以及eggNOG注释,KF-1基因组有中较多的酶参与氨基酸转运代谢、核苷酸转运代谢以及多种微生物次级代谢产物抗生素的合成。利用antiSMASH分析法在KF-1基因组中预测出22个不同的基因簇结构参与次级代谢产物的合成,有些与已知的Iturin、Fusaricidin、colistin、trdecaptin、polymyxin等抗生素基因簇的同源性很高,而另一些则尚不明确。这些具有抗菌活性的化合物分别由非核糖体肽合成酶(NPRS)、非核糖体肽合成酶联合聚酮合成酶以及Lantipeptide途径而合成。综上所述,多粘类芽孢杆菌KF-1是一种可以产生多种抗真菌活性物质的有益促生菌,可以有效地抑制多种病原真菌,具有非常广阔的应用前景。
[Abstract]:The KF-1 strain separated by our laboratory, through morphological observation, physiological and biochemical characteristics and 16S rDNA sequence analysis, KF-1 was identified as Paenibacillus polymyxa (Paenibacillus polymyxa), antibacterial experiment shows that KF-1 has a good antibacterial activity against various pathogenic fungi producing antibacterial substances. Experimental results show that the optimization of culture medium PDA is the best choice. The ethyl acetate extraction of strain KF-1 extracellular antimicrobial substances, electrospray mass spectrometry (ESI-MS) analysis showed that the above extract containing 4 groups with signal peak, antibacterial substances were: 1 peak retention time is 14.753 min, the molecular weight of 188, may be the molecular antimicrobial compound 3,5- two hydroxy benzoic acid the same amount of methyl [65]; 2 peak retention time was 22.676min, the yield is very high, and very good inhibitory effect on Gram positive bacteria and fungi were, the ion peaks of [M+Na]+ and two Double ion peak [2M+Na]+ value were 413.8803.6, which inferred that the molecular weight is 390, which belongs to macrolide antibiotic molecules; peak 3 contains two groups of compounds, molecular weight were 251 and 453, with known antibiotic molecular weight comparison, similar to the molecular weight of Polyoxin nucleoside antibiotics, its antibacterial results the compound can inhibit the growth of Candida albicans, which belongs to the antifungal antibiotics. 4 peaks including molecular weight of 882.4 and 933.5 compounds, the molecular weight of 882 LI-F and lipopeptide family Fusaridins A[M+H]+ consistent, and the molecular weight of 933 and LI-07F compounds similar. Then using whole genome shotgun. The whole genome sequence of Bacillus polymyxa KF-1 were sequenced. The results showed that KF-1 genome size is 5.1 Mb, the content of 46.26% GC, encoding 5229 ORF, which accounted for the total length of ORF base Because of the length of the 84.53% group, another 6 groups of 16S/5S/23S rRNA operon region. At the same time identified 107 non tRNA encoding, the total length of the sequence was 8275 BP, accounting for 0.001429792%. of the total length of the genome sequencing results have been submitted to the Gen Bank, the accession number is LNZF00000000. by GO injection and eggNOG release notes, KF-1 genome has more the enzymes involved in amino acid transport and metabolism, nucleotide transport and metabolism as well as a variety of microbial secondary metabolites of antibiotic synthesis. In KF-1 genome predicted 22 different gene clusters involved in the synthesis of secondary metabolites of structure by using antiSMASH analysis method, and some of the known Iturin, Fusaricidin, colistin, trdecaptin, polymyxin and other homologous antibiotic gene clusters very high, while others are not clear. These compounds with antimicrobial activity by nonribosomal peptide synthetase (NPRS), and non ribosomal peptide Enzyme synthesis combined with polyketide synthase and Lantipeptide pathway has been synthesized. In conclusion, KF-1 is a beneficial growth promoting bacterium, which can produce many antifungal substances. It can effectively inhibit various pathogenic fungi and has broad application prospects.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476

【参考文献】