O1群El Tor霍乱弧菌产毒株超级整合予的结构特征及不同培养条件下的转录谱分析

发布时间：2018-09-08 18:48

【摘要】：到目前为止,霍乱仍然是许多发展中国家面临的重要公共卫生问题。霍乱弧菌是霍乱的病原菌。1999年在El Tor霍乱弧菌N16961中首先发现了超级整合子(super-integron SI)。SI中除了少数已知的与霍乱弧菌适应性生存有关的基因外,绝大多数基因功能未明。研究提示,不同生物型霍乱弧菌SI的结构变异明显,而不同ElTor霍乱弧菌产毒株之间SI的差异是导致这些菌株之间差异的重要原因。我们采用PCR扫描结合测序的方法研究了60株1961—2008年问我国分离的霍乱弧菌产毒株的SI结构。此外,还使用实时荧光PCR的方法研究了霍乱弧菌N16961的SI中基因在M9培养基、胆盐培养基和AKI培养基中的转录情况,并与LB (luria bertani medium)的转录情况进行了比较。使用BLAST、Artemis Comparison Tool、Clustal W和MEGA 4.0等软件明确了N16961的SI中霍乱弧菌重复序列(Vibrio cholerae repeat sequence VCR)的保守和变异位点,以及VCR和基因盒的构成、分布。在VCR分析基础上,我们分析1961-2008年60株分离自中国19个省、自治区和直辖市的O1 E1 Tor霍乱弧菌产毒株SI的结构特征,绘制了这60个菌株SI内ORF (open reading frame)组成结构图。发现实验菌株SI中大部分基因盒的排列结构具有共线性的特点,但不同流行年代菌株的SI仍具有明显的多样性。多样性的表现形式包括序列插入、序列替换和序列缺失。这些变异很有可能是通过重复序列、VCR之间的重组所介导。60年代和70年代分离菌株的SI的多样性更加明显,而90年代分离菌株的SI的多样性则很低。90年代分离菌株的SI具有标志性的～24kb的片段缺失,提示不同霍乱流行高峰中分离菌株具有特征性的SI结构。实验菌株SI的结构提示,随着霍乱流行年代的变迁,流行菌株的SI结构类型呈现逐渐缩减的趋势。实验菌株中插入基因盒的序列分析提示El Tor产毒株SI基因盒可能来自其它血清群的霍乱弧菌以及拟态弧菌。 El Tor产毒株SI结构研究提示,PCR扫描结合片段测序的方法在研究El Tor产毒株SI结构具有有效性。研究结果说明不同霍乱流行中菌株的SI中的基因盒流动持续存在。将N16961在胆盐、M9和AKI培养基中培养,应用实时荧光PCR方法研究Sl内ORF的转录水平。结果显示,与在LB培养基中培养的转录结果相比,在胆盐、AKI培养条件下,少量ORF转录水平出现变化,而M9培养条件下菌株超级整合子中大部分ORF发生转录,并且表现较高的表达水平。其中少数ORF功能已知,这些功能主要集中在代谢和可移动染色体外元件,M9培养条件下还出现与DNA代谢和脂类代谢相关ORF的转录水平增高,可见SI与菌株在低营养状态下的适应性生存密切相关。
[Abstract]:Cholera remains an important public health problem in many developing countries so far. Vibrio cholerae is the pathogen of cholera. In 1999, superintegron (super-integron SI) .SI was first found in El Tor Vibrio cholerae N16961, except for a few known genes related to the adaptive survival of Vibrio cholerae. The results showed that the structural variation of SI in Vibrio cholerae was obvious, and the difference of SI among strains produced by Vibrio cholerae of different ElTor was the important reason for the difference among these strains. The SI structures of 60 strains of Vibrio cholerae isolated from 1961 to 2008 in China were studied by PCR scanning and sequencing. In addition, the transcription of SI gene of Vibrio cholerae N16961 in M9 medium, bile salt medium and AKI medium was studied by real-time fluorescence PCR, and compared with that of LB (luria bertani medium). BLAST,Artemis Comparison Tool,Clustal W and MEGA 4.0 software were used to determine the conserved and variant sites of repeat (Vibrio cholerae repeat sequence VCR) of Vibrio cholerae in N16961 SI, as well as the composition and distribution of VCR and gene cassette. On the basis of VCR analysis, we analyzed the structural characteristics of 60 strains of Vibrio cholerae O1E1 Tor produced by Vibrio cholerae O1E1 isolated from 19 provinces, autonomous regions and municipalities of China from 1961 to 2008, and plotted the composition of ORF (open reading frame) in SI of these 60 strains. It was found that the arrangement of most gene cassettes in the experimental strain SI was collinear, but the SI of the strains in different epidemic years still had obvious diversity. Diversity forms include sequence insertion, sequence substitution and sequence deletion. It is very likely that the diversity of SI in the strains isolated in the 1960s and 1970s was more evident through the recombination of repeat sequences between VCRs. However, the diversity of SI of the strains isolated in the 1990s was very low. The SI of the strains isolated in the 1990s had a signature deletion of 24kb, suggesting that the isolated strains had characteristic SI structure in different cholera epidemic peaks. The structure of the experimental strain SI suggests that with the change of cholera epidemic age, the type of SI structure of the epidemic strain is gradually decreasing. Sequence analysis of the inserted gene cassette in the experimental strain suggested that the SI gene box of El Tor producing strain might come from Vibrio cholerae of other serogroup and SI structure of Vibrio mimicus. El Tor strain. The method of sequencing is effective in studying the structure of El Tor virulent strain SI. The results showed that gene box flow in SI of different cholera strains persisted. N16961 was cultured in bile salt M9 and AKI medium, and the transcription level of ORF in Sl was studied by real-time fluorescence PCR. The results showed that, compared with the results of transcription cultured in LB medium, a small amount of ORF transcriptional level was changed under the condition of bile salt Aki culture, while most of the ORF in the super integron of strain M9 was transcripted under the condition of M9 culture. And showed a high level of expression. A few of the ORF functions have been known. These functions are mainly concentrated in the culture conditions of metabolic and removable extrinsic element M9, and the transcription level of ORF related to DNA metabolism and lipid metabolism is increased. The results showed that SI was closely related to the adaptive survival of the strain under low nutrition.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

【共引文献】