本文關鍵詞：霍亂弧菌群體感應調控因子VqmA活性相關基因的篩選及LysR和MFS蛋白功能的研究　出處：《南京農業(yè)大學》2010年碩士論文　論文類型：學位論文

  更多相關文章： 霍亂弧菌 群體感應 VqmA 多重藥物轉運蛋白 LysR

【摘要】：霍亂弧菌(V. cholerae)是一種革蘭氏陰性、兼性厭氧小桿菌,是引起人類腹瀉疾病霍亂的病原體。大量研究證明,霍亂弧菌的致病能力主要是由霍亂毒素(cholerae toxin, CT)和毒素共調節(jié)菌毛(toxin-coregulated pilus, TCP)引起的�；魜y弧菌毒力基因的表達受到各種環(huán)境條件的影響,如營養(yǎng)物濃度和種類、溫度、pH、膽汁鹽和群體感應信號分子。 HapR是霍亂弧菌群體感應調控系統(tǒng)的關鍵調控因子,可以抑制霍亂弧菌CT和TCP表達。LuxR家族蛋白VqmA可以在低細胞濃度時誘導HapR的表達從而降低細菌的致病性。本文通過構建轉座子隨機突變文庫的方法篩選影響VqmA活性的基因,獲得七株候選的突變株,并且對突變株M1(△VC0073, SAM-依賴的甲基轉移酶)做了深入的研究。研究發(fā)現(xiàn)該基因缺失后,VqmA無法激活報告基因的表達,并且VqmA的轉錄調控活性隨著該基因的回復表達而恢復,VC0073是VqmA發(fā)揮活性的必須基因。VC0073編碼合成的蛋白與SAM-依賴的甲基轉移酶的同源性最高,在很多細菌中該酶參與一些小分子化合物的合成,VqmA激活目的基因的過程可能需要該酶合成的某類化合物的協(xié)助,但是研究發(fā)現(xiàn)該化合物并不存在于細菌上清液中。 LysR類蛋白是一種全局性的轉錄調控因子,霍亂弧菌中約有40個LysR類型的蛋白。借助生物信息學工具,在霍亂弧菌中發(fā)現(xiàn)了五類比較特殊的LysR蛋白,這些蛋白的下游都為MFS家族蛋白。MFS家族蛋白屬于多重藥物轉運蛋白。我們選取其中一組基因VC1617 (Encoding LysR Protein)和VC1618 (Encoding Multidrug Resistance protein)作為研究對象,研究發(fā)現(xiàn)VC1617缺失和過表達后霍亂弧菌可以在LB和M9培養(yǎng)基正常生長,VC1617并不是細菌在這兩中培養(yǎng)基生長的必需基因,這在一定程度上也反映出了LysR蛋白生理功能的多樣性。通過構建Luminescence檢測質粒發(fā)現(xiàn)LysR不但可以抑制自身的表達而且還可以激活下游基因VC1618 (Multidrug Resistance Gene)的表達。Western blot試驗表明VC1617缺失后霍亂弧菌毒力基因的表達并沒有受到影響；雖然VC1 617缺失后多重藥物轉運蛋白VC1618的表達量大為降低,但是突變株對一些特定藥物的耐受能力并沒有削弱；在毒力基因的表達量和藥物的耐受能力都沒有降低的情況下,突變株在小鼠體內的定殖能力也沒有降低。
[Abstract]:Vibrio cholerae (V. cholerae) is a gram negative, facultative anaerobic bacteria is small, causing cholera diarrhea disease human pathogens. Many studies have demonstrated that Vibrio cholerae pathogenicity is mainly caused by cholera toxin (cholerae toxin, CT) and the toxin coregulated pilus (toxin-coregulated pilus, TCP). The influence of expression caused by Vibrio Cholera toxin genes are subjected to various environmental conditions, such as the type and concentration of nutrients, temperature, pH, bile salt and quorum sensing signal.
HapR is a key regulator of V.cholerae quorum sensing regulation system, can inhibit Vibrio cholerae CT and TCP expression of.LuxR family protein VqmA can induce HapR cells at low concentration so as to reduce pathogenic bacteria. The construction method of transposon random mutation library screening influence the activity of VqmA gene, seven strains of candidate the mutant strain, and the mutant M1 (VC0073, SAM- dependent methyl transferase) were studied. The study found that the gene deletion, VqmA could not activate the reporter gene expression, transcriptional regulation and VqmA control activity restored along with the expression of this gene is VqmA VC0073 reply, the activity must play methyl.VC0073 protein and SAM- gene encoding synthesis dependent transferase homology in many bacteria, the enzymes involved in some small molecular compounds synthesis, activation of VqmA gene The process may require the assistance of some of the compounds synthesized by the enzyme, but the study found that the compound does not exist in the bacterial supernatant.
LysR protein is a global transcriptional regulator, Vibrio cholerae has about 40 types of LysR protein. Using bioinformatics tools, five kinds of special LysR protein was found in Vibrio cholerae, these proteins are downstream of the MFS protein family.MFS family proteins belong to multiple drug transporters. We selected a group of genes VC1617 (Encoding LysR Protein) and VC1618 (Encoding Multidrug Resistance protein) as the research object, the study found that deletion of VC1617 and overexpression of Vibrio cholerae can grow normally in the culture medium of LB and M9, VC1617 is not the genes required for the growth of bacterial culture in two, which to some extent also reflects the diversity of LysR protein physiological function. By constructing the Luminescence plasmid detection found LysR not only inhibits its own expression but also can activate downstream gene VC1618 (Mu Ltidrug Resistance Gene.Western blot) expression test showed that the expression of virulence genes of Vibrio cholerae VC1617 deletion was not affected; while the expression of VC1 617 after deletion of multiple drug transporter VC1618 is greatly reduced, but the mutant for certain drug resistance ability has not weakened; in the expression of virulence genes and the amount of drug tolerance has been reduced under the condition of the mutant colonization on the mice are not reduced.

【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378

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本文編號：1384299