【作者】 姚帆；

【导师】 关雄；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 细菌能通过信息分子的浓度来感知其数量,从而诱发一个从量变到质变的过程,这其中受到群体感应系统(quorum-sensing,简称QS)调节机制的调控。N—乙酰高丝氨酸内酯(N-acyl-homoserine lactones,AHLs),是一类广泛存在的革兰氏阴性细菌群体密度感应系统中的信号分子,参与诱导调控许多植物病原菌致病基因的表达。由aiiA基因编码的AiiA蛋白能降解这类AHLs分子,阻断细菌群体密度感应系统,从而减弱利用QS系统的病原菌的产生的危害。越来越多的研究报道了芽孢杆菌属(Bacillus)中分布有AiiA蛋白。本研究设计一对简并引物aiiAl(5′-ATG ACA GTA AAR AAR CTT TAT TTC-3′)和aiiA2(5′-CTA TAT ATA YTCHGG GAA CRC TYT A-3′),利用PCR检测法初步研究了aiiA基因在枯草芽孢杆菌(Bacillus subtilis,简称Bs)中的分布情况。实验结果表明:有50%左右的检测菌种分布有aiiA基因,本研究首次发现了类似aiiA的基因在Bs的分布。从中选取了来源于Bs-2的aiiA基因进行分子克隆实验,对aiiA基因进行一系列生物信息学分析,此序列与其他在GenBank上登录的序列同源性高达95%,随后通过SWISS-MODEL模拟出它的蛋白三维结构,构建系统进化树。把aiiA-B2基因克隆到pET-29a表达载体中,构建重组质粒pETB2,IPTG诱导后可大量表达目的蛋白。致病性检测表明该BS-2来源的AiiA蛋白不仅能很好地降解OHHL信号分子,而且对胡萝卜欧文氏软腐病菌具有较强的抗病作用。本研究的完成不但有助于了解Bs的防病基因,还为深入了解AiiA蛋白的作用机理奠定基础。更多还原

【Abstract】 Quorum sensing is a mechanism in which bacteria coordinate the expression of certain genes in response to their population density by producing, releasing and detecting signal molecules called autoinducers. Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. AiiA is a protein which can block the bacterial quorum sensing by hydrolyzing AHL-lactone, and could greatly attenuate the disease caused by many bacterial pathogens in which quorum sensing regulate the expression of virulence genes. In this study, a pair of primers were designed from the conserved sequences of aiiA gene in the genomes of several Bacillus strains, and the gene was amplified from Bacillus subtilis BS-2 by PCR. AiiA gene was cloned to a cloning vector pMD18-T and sequenced. The open reading frame of the aiiA gene was 753 bp, and the similarity of aiiA gene from B. subtilis BS-2 to other sequences in the GenBank was as high as 95%. This gene was subcloned into an expression vector pET29a, and the recombinant AiiA protein was overexpressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed AiiA protein was estimated to be 28 kDa by SDS - PAGE. The lysate of the recombinant E. coli could not only repress the pigment synthesize of the quorum sensing system reporter strain JAZ1, but also attenuate the effect of soft rot symptoms. This study was important for further research on the disruption of infections caused by Gram-negative bacteria in agriculture.更多还原