本文關(guān)鍵詞：兩株肉毒梭菌（A str.230611，，F(xiàn) str.230613）全基因組測序分析及pH環(huán)境因素對毒素基因表達(dá)的影響、調(diào)控研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肉毒梭菌 基因組 毒素基因簇 調(diào)控基因botR 共表達(dá) 正調(diào)控

【摘要】：肉毒梭菌(C.botulinum)是一組能產(chǎn)生肉毒毒素的細(xì)菌的統(tǒng)稱。它們是一群專性厭氧的,短桿狀革蘭氏陽性菌。這些細(xì)菌屬于梭菌屬中不同的種,并且分為4個(gè)群。 近年來越來越多的研究證明肉毒梭菌可以通過腸道感染致病,但是對于其粘附,侵入并定殖腸道的致病機(jī)理了解較少。肉毒毒素是肉毒梭菌致病性的重要方面。肉毒毒素的編碼基因存在于一個(gè)15kb大小的毒素基因簇上。目前對于這個(gè)毒素基因簇的進(jìn)化來源,不同型毒素基因簇的插入位點(diǎn)差異及插入規(guī)律規(guī)律,毒素基因簇結(jié)構(gòu)多樣性產(chǎn)生機(jī)制的認(rèn)識比較少。為了解決以上問題,本研究測定了A型(230611)和F型(230613)肉毒梭菌全基因組序列,并且對基因組結(jié)構(gòu)、基因組穩(wěn)定性、肉毒梭菌進(jìn)化分類、肉毒梭菌腸道感染相關(guān)基因、毒素基因簇序列及排列、毒素基因簇重組事件等進(jìn)行分析和比較。 肉毒梭菌腸道致病是近年來發(fā)現(xiàn)的一個(gè)條件性致病現(xiàn)象,對于其在人體消化道不同環(huán)境中的適應(yīng)性及毒素基因表達(dá)調(diào)控研究較少。肉毒梭菌通過人體消化道各器官時(shí)會(huì)經(jīng)歷酸性、中性和堿性環(huán)境。細(xì)菌在產(chǎn)毒方面對pH值的改變會(huì)作出相應(yīng)變化,調(diào)控自身基因表達(dá)量以適應(yīng)環(huán)境并保持致病性。目前認(rèn)為毒素基因簇中botR基因是毒素基因表達(dá)的調(diào)控基因,但是對于在細(xì)菌內(nèi)部實(shí)際生理狀態(tài)下botR基因?qū)Χ舅鼗騜ont的調(diào)控作用及轉(zhuǎn)錄量變化關(guān)系還缺乏系統(tǒng)的研究。 為了深入了解肉毒梭菌對人體消化道不同pH環(huán)境的適應(yīng)性,毒素基因表達(dá)的變化及調(diào)控基因botR對毒素基因bont的調(diào)控作用,我們挑選毒力較強(qiáng)的A型(230611)肉毒梭菌在不同pH環(huán)境下培養(yǎng),測定pH值對細(xì)菌生長、毒素基因和調(diào)控基因轉(zhuǎn)錄量變化及相關(guān)性、毒素蛋白分泌量的差別,從而探索毒素基因表達(dá)的調(diào)控機(jī)制。 第一部分A(230611)、F(230613)型肉毒梭菌全基因組序列測定、進(jìn)化分類、基因組穩(wěn)定性及致病基因分析 我們使用第二代高通量測序技術(shù)(Roche 454 GS)測定了A型(230611)和F型(230613)肉毒梭菌全基因組序列。測序深度分別為全基因組的13.7倍和10.1倍,覆蓋率均為99%以上。根據(jù)參考序列設(shè)計(jì)引物測定缺口序列,得到基因組完整的序列。使用16S rRNA聚類法對已完成全基因組測序的肉毒梭菌菌株進(jìn)行進(jìn)化分類,結(jié)果顯示對第一群(Group I)內(nèi)部菌株的聚類結(jié)果可信度較低。我們采用類似MLST的方法對相同菌株進(jìn)行聚類,聚類結(jié)果能正確反映菌株的進(jìn)化關(guān)系,并且能精確區(qū)分A1亞型菌株內(nèi)部的進(jìn)化關(guān)系。聚類結(jié)果表明,A(230611)菌株屬于第一群的A1亞型菌株,在四個(gè)A1亞型菌株中最早分化;F(230613)型菌株屬于第一群F型菌株,與F str. Langeland菌株進(jìn)化關(guān)系最近。 分析肉毒梭菌基因組中的移動(dòng)元件結(jié)果顯示,基因組中均不含有完整的插入序列(IS),只有6個(gè)和5個(gè)殘余的插入序列(partial IS)。使用密碼子偏愛性分析基因組島(Genomic Island, GI),結(jié)果顯示兩個(gè)菌株不含基因組島。以上結(jié)果表明A(230611)、F(230613)型菌株基因組比較穩(wěn)定,可能與其生活周期長期處于芽孢休眠狀態(tài)有關(guān)。致病基因分析表明A(230611)、F(230613)型肉毒梭菌基因組中各有9個(gè)和11個(gè)與細(xì)菌粘附和侵入相關(guān)的基因,這些基因包含粘附素(intimin)、侵襲素(invasin)、鞭毛鉤相關(guān)蛋白(Flagellar hook-associated)等結(jié)構(gòu)域,并且在同型的其它菌株中也存在這些基因,可能與肉毒梭菌腸道感染的粘附侵入有關(guān),下一步可以通過實(shí)驗(yàn)證明這些基因的功能。 與最近源物種且不產(chǎn)肉毒毒素的生孢梭菌基因組比對及GC含量分析表明,在進(jìn)化過程中,A(230611)、F(230613)型肉毒梭菌在基因組特定位置通過水平基因轉(zhuǎn)移方式,攝入一段外來的含有肉毒毒素基因簇的片段,從而獲得產(chǎn)生肉毒毒素的能力。同時(shí)比對生孢梭菌與A1,A2、F型(230613)肉毒梭菌基因組,我們發(fā)現(xiàn)三個(gè)肉毒梭菌的毒素基因簇分別插入染色體不同位點(diǎn)。通過基因組比對,我們將9個(gè)肉毒毒素基因簇的插入位點(diǎn)分別定位到F型(230613)菌株基因組的三個(gè)位點(diǎn)。插入位點(diǎn)分析表明,肉毒毒素基因簇片段的插入不是隨機(jī)發(fā)生的,而是有規(guī)律地插入染色體上3個(gè)位點(diǎn)之一。A1、B型毒素基因簇傾向于插入site 3,A2型傾向于site 1,F型(230613)傾向于site 2。各型毒素基因簇結(jié)構(gòu)比較表明,非毒素基因部分具有位點(diǎn)特異性,而毒素基因流動(dòng)性較大。我們發(fā)現(xiàn)site 2位點(diǎn)的bontF毒素基因簇與site 3位點(diǎn)的bontA1毒素基因簇發(fā)生了重組事件,形成新的bontA1毒素基因簇結(jié)構(gòu)。這也是肉毒毒素基因簇多樣性的形成機(jī)制。 第二部分pH值對A型(230611)肉毒梭菌毒素基因bontA表達(dá)的影響及調(diào)控基因botR的調(diào)控作用 肉毒梭菌在人體腸道感染過程中會(huì)經(jīng)歷酸性,中性和堿性pH環(huán)境。為了深入了解A型(230611)肉毒梭菌在產(chǎn)毒方面對不同pH環(huán)境的適應(yīng)性,我們系統(tǒng)性地測定不同pH(6.0,7.0,8.0,9.0)條件下細(xì)菌不同生長時(shí)間點(diǎn)bontA基因的轉(zhuǎn)錄量,以及毒素蛋白分泌量。為了研究毒素基因簇中調(diào)控基因botR對毒素基因bontA的調(diào)控作用,我們同時(shí)測定botR基因的轉(zhuǎn)錄量,并且研究bontA與botR基因轉(zhuǎn)錄量變化的相關(guān)性。我們進(jìn)一步對不同環(huán)境條件下不同生長期的細(xì)菌培養(yǎng)物進(jìn)行轉(zhuǎn)錄組RNA測序,從而深入探究毒素基因表達(dá)調(diào)控機(jī)理。 在轉(zhuǎn)錄水平,針對毒素基因bontA轉(zhuǎn)錄量測定結(jié)果顯示:pH 7.0條件下轉(zhuǎn)錄量最高,酸性或堿性條件下轉(zhuǎn)錄量顯著降低(P0.01),pH 9.0條件下轉(zhuǎn)錄量最低(P0.01)。提示細(xì)菌為了適應(yīng)酸性和堿性環(huán)境,會(huì)在轉(zhuǎn)錄水平抑制毒素基因bontA的表達(dá)。在蛋白水平,針對毒素蛋白分泌量測定結(jié)果顯示:pH 7.0條件下毒素蛋白分泌量最高,pH 6.0和pH 8.0條件下分泌量降低,與毒素基因轉(zhuǎn)錄被抑制有關(guān)。但是pH9.0條件下毒素蛋白分泌與pH7.0條件下無明顯差別。推測原因?yàn)?雖然pH值9條件下bontA基因的轉(zhuǎn)錄受到抑制,但是轉(zhuǎn)錄后的翻譯或轉(zhuǎn)運(yùn)效率增高。 調(diào)控基因botR轉(zhuǎn)錄量測定結(jié)果顯示:pH 7.0條件下botR基因轉(zhuǎn)錄量最高,酸性或堿性條件下轉(zhuǎn)錄量降低(P0.05),pH 9.0條件下轉(zhuǎn)錄量最低(P0.01)。botR基因與bontA基因轉(zhuǎn)錄量相關(guān)性分析表明:不同pH條件下botR基因和bontA基因轉(zhuǎn)錄量在生長過程中是正相關(guān)的,相關(guān)系數(shù)介于0.7和1.0之間。這說明botR基因是bontA基因的正調(diào)控基因,在細(xì)菌生長周期,bontA基因轉(zhuǎn)錄量與botR基因轉(zhuǎn)錄量協(xié)同變化。 我們選取毒素基因轉(zhuǎn)錄量差別較大的pH7和pH9兩個(gè)條件培養(yǎng)細(xì)菌,分別取指數(shù)期、穩(wěn)定期和衰亡期的菌液進(jìn)行轉(zhuǎn)錄組測序,準(zhǔn)備通過共表達(dá)基因聚類分析來尋找與毒素基因表達(dá)相關(guān)的轉(zhuǎn)錄因子,順式作用元件,以及調(diào)控RNA,如5’UTR, 3’UTR, small RNA等。
[Abstract]:Clostridium botulinum (C.botulinum) is a group of bacteria that can produce botulinum toxin. They are a group of specialized anaerobic, short rod gram positive bacteria. These bacteria belong to different species of Clostridium, and they are divided into 4 groups.
In recent years, more and more studies showed that Clostridium botulinum can through the intestinal infection, but the adhesion, invasion and colonization of the intestinal pathogenic mechanism. Little is known about the botulinum toxin is an important aspect of the pathogenicity of Clostridium botulinum. Gene encoding botulinum toxin present in the toxin gene cluster size on a 15KB. At present, the toxin gene cluster evolutionary origin, insertion and the regularity of the insertion sites of different type of toxin gene cluster, less toxin gene cluster structure diversity mechanism. In order to solve the above problems, A type were determined in this study (230611) and F (230613) whole genome sequence of Clostridium botulinum. The genome structure, genome stability, evolutionary classification of Clostridium botulinum, intestinal infection related gene of Clostridium botulinum toxin gene cluster, and sequence alignment, toxin gene cluster recombination events were analyzed and compared.
Clostridium botulinum is a conditional pathogenic intestinal pathogenic phenomenon discovered in recent years, in the human digestive tract in different environment adaptability and toxin gene expression research. Clostridium botulinum through the digestive organ of the human body will experience acidic, neutral and alkaline environment. The value of pH in the bacterial toxin producing the change will make the corresponding change, to adapt to the environment and keep the pathogenic expression of their genes. The regulation of botR toxin gene cluster genes are toxin gene expression, but the change of regulation and the relationship between the amount of transcription of BoNT gene from the lack of systematic research on bacterial botR gene internal actual physiological condition.
In order to understand the adaptability of Clostridium botulinum on human digestive tract in different pH environment, role change and expression of botR toxin gene on the expression of BoNT toxin gene, we selected strong virulent type A (230611) of Clostridium botulinum in different pH environment culture, determination of pH value on the growth of bacteria, the gene expression quantity the toxin gene and regulation of toxin protein secretion and the correlation between the quantity difference, so as to explore the regulation mechanism of toxin gene expression.
The first part of A (230611), F (230613) type of Clostridium botulinum genome sequencing, evolutionary classification, genomic stability and pathogenic gene analysis
We use the second generation high-throughput sequencing technology (Roche GS 454) A was determined (230611) and F (230613) whole genome sequence of Clostridium botulinum. The sequencing depth of whole genome were 13.7 times and 10.1 times, the coverage rate was more than 99%. According to the measured gap sequence reference sequences obtained. The complete genome sequence of Clostridium botulinum strains. The whole genome sequencing has been completed by phylogenetic classification using 16S rRNA clustering method, the results showed that the first group (Group I) clustering results of internal strains with low confidence. We use the method of MLST on the same strain clustering, clustering results can correctly reflect the phylogenetic relationship of strains, and can accurately distinguish the evolutionary relationship between internal A1 subtype strains. The clustering results showed that A (230611) strains belonging to the first group of subtype A1 strains, the earliest differentiation in the four subtype A1 strains; F (230613) strains belong to the A group of F strains are closely related to the evolution of the F str. Langeland strain.
Analysis of the moving element of Clostridium botulinum genome showed that the genome does not contain complete insertion sequence (IS), only 6 and 5 of residual inserted sequence (partial IS). Using the codon preference analysis of genomic island (Genomic Island, GI), the results showed that two strains containing genomic island. These results showed that A (230611), F (230613) strain genome is relatively stable, and the life cycle of long-term in a state of dormancy. Bacillus virulence gene analysis showed that A (230611), F (230613) of 9 and 11 with bacterial adhesion and invasion related genes in the genome of Clostridium botulinum type and these genes contain adhesin (intimin), invasin (invasin), flagellar hook associated protein (Flagellar hook-associated) and other domains, and these genes also exist in other strains of the same type, may be related to the adhesion of Clostridium botulinum invasion of intestinal infection, The next step can be proved by experiments to prove the function of these genes.
And c.sporogenes genome alignment and the content of GC and recent source species does not produce botulinum toxin analysis shows that in the process of evolution, A (230611), F (230613) of Clostridium botulinum type in a specific location in the genome through lateral gene transfer, intake of a foreign gene fragment containing botulism clusters. In order to produce botulinum toxin of Clostridium sporogenes. At the same time compared with A1, A2, F (230613) of Clostridium botulinum toxin genome, we found three gene clusters of Clostridium botulinum were inserted into the chromosome of different loci. By genome comparison, we will insert the 9 loci of botulinum toxin gene cluster respectively. Type F (230613) of three loci. The genomic insertion site analysis showed that botulinum toxin gene cluster insert is not random, but regularly inserted into one of the 3 loci on chromosome.A1, B toxin gene cluster tend to plug In site 3, A2 tend to site 1, F (230613) tend to site 2. different types of toxin gene cluster structure shows that the non toxin gene with site-specific, and toxin gene mobility. We found that the bontF toxin gene cluster and site site 2 loci of bontA1 toxin gene recombination events the 3 cluster sites, the formation of bontA1 toxin gene cluster structure. The formation mechanism of the diversity of the botulinum toxin gene cluster.
The effect of the second part pH on the expression of A (230611) Clostridium botulinum toxin gene bontA and the regulation of the regulatory gene botR
Clostridium botulinum will experience acid in human intestinal infection in neutral and alkaline environment of pH. In order to understand the type of A (230611) of Clostridium botulinum toxin production of pH in different environments, we systematically measured pH (6.0,7.0,8.0,9.0) transcription of bacteria at different long time under the condition of bontA gene well, the secretion of toxin protein. In order to control the role of regulation of the toxin gene cluster in botR gene of bontA toxin gene, we also measured transcription of the botR gene, and the correlation between changes of bontA and botR mRNA. We further transcriptome sequencing of RNA culture in different growth periods of bacteria in different environmental conditions thus, in-depth study of toxin gene expression regulation mechanism.
At the transcriptional level, according to the bontA gene transcript levels indicated that transcription of pH 7 under the condition of the highest transcript levels in acidic and alkaline conditions significantly decreased (P0.01), transcription was the lowest under the condition of pH 9 (P0.01). It is suggested that bacteria in order to adapt to the acid and alkaline environment, can inhibit the expression of bontA gene at the transcriptional level at the protein level, the toxin protein secretion measured results showed that the toxin protein pH 7 under the condition of the secretion of the highest, pH 6 and pH 8 under the condition of reduced secretion, associated with toxin gene transcription is inhibited. But under the condition of pH9.0 toxin protein secretion and pH7.0 under the condition of no significant difference. The possible reason is: Although the pH value under the condition of 9 bontA gene transcription was inhibited, but after transcription translation or transfer efficiency increased.
Results show that the determination of botR gene transcription: transcription of botR gene in pH 7 under the condition of the highest transcript levels in acidic and alkaline conditions (P0.05), reduce the minimum quantity of transcription under the condition of pH 9 (P0.01) of.BotR gene and bontA gene expression correlation showed that the botR gene and bontA gene expression under different pH conditions is positive correlation in the growth process, the correlation coefficient between 0.7 and 1. This indicates that botR gene is the bontA gene, the growth cycle in bacteria, bontA gene transcription of botR gene transcription and the amount of collaborative change.
We selected a large quantity of toxin gene transcription of pH7 and pH9 two cultured bacteria, were obtained from the exponential phase, stationary phase and decline phase of bacteria were prepared by transcriptome sequencing, gene cluster analysis to find the co expression of transcription factors associated with toxin gene expression, cis acting elements, and the regulation of RNA, such as 5 'UTR, 3' UTR, small RNA and so on.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

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