温和气单胞菌强弱毒株全基因组重测序及体外抑菌效应研究

发布时间：2018-06-20 01:57

本文选题：温和气单胞菌 + 强弱毒株　；参考：《吉林农业大学》2017年硕士论文

【摘要】：温和气单胞菌在自然界中分布广泛,是人兽共患的机会性致病菌。当机体免疫力低下时,温和气单胞菌易从潜伏菌变为致病菌,并且使用大量的抗菌素易使菌株变为耐药菌。本研究从病死鱼中分离出2株温和气单胞菌,通过动物回归试验检测其毒株强弱;通过Illumina HiSeq 2500测序仪将提取DNA构建的文库进行全基因组重测序,并利用COG、GO数据库对比分析2株菌的基因差异;通过全基因组重测序寻找CRISPR位点并进行耐药机制检测;通过5种常用水产药物检测2株温和气单胞菌体外抑菌浓度并确定最佳抑菌时间。为温和气单胞菌的致病机制及耐药机制提供科学依据。其实验结果如下:1动物回归试验检测结果为,A1菌半致死浓度为2.7×105 cfu/0.5 mL;B2菌半致死浓度为1.6×105 cfu/0.5 mL。2 A1、B2蛋白编码区域的SNP中,77%是同义编码突变,23%非同义编码突变。3 2株菌株中有24293个核苷酸为非同义突变。A1发生的非同义突变基因数目为2492,B2发生的非同义突变基因数目为2488。4 A1、B2所产生的差异基因突变个数分别为2564、2565。5 B2突变基因位置存在于gene rna1865位,这也使得在统计COG数据时,碳水化合物转运与代谢[G],A1为144个,B2为145个。6编码rna 1865位基因的碱基一共有135个,一共有107个SNPs基因位点发生突变,有1个基因发生非同义编码突变。7与参考基因组比较,在参考序列位点坐标第27407,T碱基突变为A碱基。8 2株温和气单胞菌中可信CRISPR个数为1,总长度为2805bp;42条间隔序列;可疑CRISPR个数为3,总长度为384bp。对可信CRISPR的重复序列二级结构进行预测表明,头环由15个碱基构成;茎环含有5个碱基,总体形成发夹环。在进行BLAST比对时显示,除其他同属或同源种属外,DR与嗜热螺旋体DSM 6192、暗网菌属等远缘菌种均有较高的同源性,且置信值为1。9 5种常用水产药物对2株温和气单胞菌均能产生PAE,8 x MIC的作用时间最短、16 x MIC和32 x MIC作用的时间最长。这均与已研究的结果相符。并且低浓度的服乐兴和恩诺杀星均能对产生较长的PAE。
[Abstract]:Aeromonas mildii, which is widely distributed in nature, is the opportunistic pathogen of zoonosis. When immunity is low, Aeromonas mildii can easily change from latent bacteria to pathogenic bacteria, and the use of a large number of antibiotics can easily turn the strains into drug-resistant bacteria. In this study, two strains of Aeromonas mildifolia were isolated from diseased and dead fish, and their virulence was detected by animal regression test, and the library constructed by Illumina HiSeq 2500 was sequenced by Illumina HiSeq 2500 sequencer. The gene differences of the two strains were compared with COGG go database, and the CRISPR loci were found by genome re-sequencing and the drug resistance mechanism was detected. In vitro inhibitory concentration of 2 strains of Aeromonas mildii was detected by 5 kinds of commonly used aquatic drugs and the optimum bacteriostatic time was determined. It provides scientific basis for pathogenic mechanism and drug resistance mechanism of Aeromonas mildii. The results of the experiment are as follows: the result of the animal regression test is as follows: the semi-lethal concentration of A1A1 is 2.7 脳 105mLLB2, and the semi-lethal concentration of SNPs is 1.6 脳 105mL.2 A1OB2 protein coding region. 77% of the SNPs are synonymous coding mutations and 23.3% non-synonymous coding mutations. 2 strains of SNPs. There were 24293 nonsynonymous mutation genes with 2 4293 nucleotide nonsynonymous mutations. The number of non-synonymous mutations was 2492B 2. The number of non-synonymous mutation genes was 2488.4 A1nb 2. The number of differential gene mutations was 2564% 2565.552 B 2 mutation in the gene rna1865 locus, respectively. This also resulted in a total of 135 bases encoding 145 rna 1865 position genes in carbohydrate transport and metabolism [G] A 1 = 144 B 2 and 145 rna 1865 position genes, and 107 SNPs gene loci were mutated. Compared with the reference genome, the number of credible CRISPR was 1 and the total length of CRISPR was 2 805bp2 in the reference sequence coordinate 27407 T base mutation to A base 8.82 strain. The number of suspected CRISPR was 3 and the total length was 384bp. The secondary structure of the repeat sequence of trusted CRISPR was predicted. The head ring was composed of 15 bases, and the stem ring contained 5 bases, forming a hairpin ring as a whole. The comparison of blast showed that there was a high homology between Dr and DSM61922, except for other genus or homologous species, and the other distant species, such as the genus DSM61922, had a high homology with the genus DSM61922, and there was a high homology between Dr and DSM61922. The confidence value was that 1.95 common aquatic drugs could produce PAE 8 x MIC with the shortest time of 16 x MIC and 32 x MIC. These results are in good agreement with the results obtained. And low concentration of Lexing and enroxide can both produce a longer PAE.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S941.4

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