本文關鍵詞：多粘類芽孢桿菌KF-1抗真菌物質(zhì)的純化及其編碼基因簇分析　出處：《濟南大學》2016年碩士論文　論文類型：學位論文

  更多相關文章： 多粘類芽孢桿菌 抑菌物質(zhì) 高效液相色譜 電噴霧質(zhì)譜 基因簇

【摘要】：菌株KF-1由本實驗室分離,通過形態(tài)觀察、生理生化特征以及16S rDNA序列分析將KF-1鑒定為多粘類芽孢桿菌(Paenibacillus polymyxa),抑菌實驗顯示KF-1對多種病原真菌具有很好的抑菌活性。產(chǎn)抑菌活性物質(zhì)培養(yǎng)優(yōu)化實驗結果顯示PDA培養(yǎng)基為最佳選擇。進一步采用乙酸乙酯法萃取菌株KF-1胞外抑菌活性物質(zhì),電噴霧質(zhì)譜法(ESI-MS)分析顯示以上萃取物中含4組具有抗菌活性物質(zhì)的信號峰,分別為:峰1保留時間為14.753 min,分子量為188,可能為抑菌化合物3,5-二羥基苯甲酸甲酯[65]的分子量一致;峰2保留時間為22.676min,產(chǎn)量非常高,并且對革蘭氏陽性菌以及真菌均有著很好的抑制效果,其離子峰[M+Na]+及二倍離子峰[2M+Na]+質(zhì)荷比分別為413.8、803.6,從而推斷該分子量為390,屬于大環(huán)內(nèi)酯類小分子抗生素;峰3主要包括兩組化合物,分子量分別為251和453,通過與已知抗生素分子量比較,與核苷類抗生素Polyoxin的分子量相類似,其抑菌結果顯示該化合物可抑制白色念珠菌的生長,屬于抗真菌類抗生素。峰4主要包括分子量為882.4以及933.5的化合物,其分子量為882與LI-F脂肽家族中的Fusaridins A[M+H]+相一致,而933分子量則與LI-07F化合物類似。接下來利用全基因組鳥槍法,對多粘類芽孢桿菌KF-1的全基因組序列進行了測序分析。結果顯示:KF-1基因組大小為5.1 Mb,GC含量46.26%,共編碼5229個ORF,其中ORF總長度占基因組長度的84.53%,另外有6組16S/5S/23S rRNA操縱子區(qū)域。同時鑒定了107個非編碼tRNA,其序列總長度為8275 bp,占基因組總長度的0.001429792%�；蚪M測序結果已提交到Gen Bank,登錄號為LNZF00000000。通過GO注釋以及eggNOG注釋,KF-1基因組有中較多的酶參與氨基酸轉(zhuǎn)運代謝、核苷酸轉(zhuǎn)運代謝以及多種微生物次級代謝產(chǎn)物抗生素的合成。利用antiSMASH分析法在KF-1基因組中預測出22個不同的基因簇結構參與次級代謝產(chǎn)物的合成,有些與已知的Iturin、Fusaricidin、colistin、trdecaptin、polymyxin等抗生素基因簇的同源性很高,而另一些則尚不明確。這些具有抗菌活性的化合物分別由非核糖體肽合成酶(NPRS)、非核糖體肽合成酶聯(lián)合聚酮合成酶以及Lantipeptide途徑而合成。綜上所述,多粘類芽孢桿菌KF-1是一種可以產(chǎn)生多種抗真菌活性物質(zhì)的有益促生菌,可以有效地抑制多種病原真菌,具有非常廣闊的應用前景。
[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.

【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S476

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