AHL分子荧光法检测质粒的构建.docVIP

AHL分子荧光法检测质粒的构建 摘要：群体感应是细菌生长到一定密度时相互感应，并进行基因表达及调控产生的独特、多样的群体行为现象。N-酰基高丝氨酸内酯（N-acyl-homoserine lactones，AHL）类化合物是革兰阴性菌群体感应中最重要的一类信号分子，调控许多生理特性基因的表达。快速、简便、有效地检测细菌能否产生AHL或产生何种信号分子，成为深入研究和了解细菌群体感应的重要手段。本论文试图构建一个含有绿色荧光蛋白基因的生物传感器，通过该生物传感器在含AHL的培养液中的荧光产生情况来检测环境中的AHL是否达到值域。试验方法主要使用pLuxGFPuv2和pLuxR两种质粒通过PCR扩增，酶切和拼接等操作构建含有GFP基因的检测质粒并转化宿主，观察biosensor在含AHL的培养基中的产荧光情况。4509 关键词： 群体感应；AHL信号分子；生物传感器；检测Construction of a plasmid for detecting Acyl-homoserine lactones Abstract: Quorum sensing is the unique,diverse group behavior phenomenon occurred when the bacterium grows to a certain density and responses mutually,and it is responsible for the gene expression and the regulation. Many gram-negative bacteria are known to use acylhomoserine lactones (acyl-HSLs) as intercellular signals, called autoinducers, in density dependent gene expression, autoinducers at high cell densities can achieve critical concentration and activate the expression of specific genes. Rapid, simple and efficient detection of bacteria wether can produce signal molecules AHL or other kinds of signal molecules, become important means of in-depth study and understand of bacterium quorum sensing. Biosensor method was used in this paper attempted to detect whether the signal concentration reaches a threshold level. We chose plasmid pLuxGFPuv2 and plasmid pLuxR, using ways of PCR, enzyme digestion and ligation to construct a novel lasmid which contains GFP. The plasmid was than transformed into a host and the case of the biosensor producing fluorescence can be watched. 不同的细菌在繁殖过程中分泌不同的信号分子来调控基因的表达，这种信号分子利用信号分子在细胞间扩散，来感知自身和周围环境中其他菌群的数量变化，当信号分子浓度达到一定阈值时就启动菌体相关基因表达，调控相关生物学功能[5]。如产生毒素、形成生物膜、产生抗生素、生成孢子、产生荧光等，以适应环境的变化，我们将这一现象称为群体感应调节（quorum sensing，QS）。这一感应现象只有在细菌密度达到一定阈值后才会发生，所以也有人将这一现象称为细胞密度依赖的基因表达（cell density de-pendent control of gene expression）。据已有的研究表明，革兰氏阴性菌通过产生高丝氨酸内酯类 (acylmdash;homoserine lactonase，AHLs)作为细胞间的信号分子，该信号分子以细胞密度依赖的方式调控生理特性的表达；革兰氏阳性菌利用一些小肽作为QS的信号分子来感知种内自身种群数量，协调多种基因的表达；还有一类信号分子是一种呋喃酰硼酸二酯，即自体