群体感应信号降解酶产生菌的筛

发布时间：2018-06-24 08:05

本文选题：群体感应淬灭 + 酰基高丝氨酸内酯　；参考：《扬州大学》2017年硕士论文

【摘要】：群体感应(quorum sensing,QS)是一种细菌细胞间信号分子的传递机制,即细菌可以通过感应自身的自体诱导分子(autoinducer,AI),如N-乙酰高丝氨酸内酯(AHLs)的浓度来协调细菌的相关基因的表达以此来适应多变的自然环境。许多植物病原细菌通过群体感应系统来调控毒性基因的表达,因此群体感应系统是潜在的植物病害防治靶标。这种对细菌QS调控机制的干扰和破坏称为群体感应淬灭(quorum quenching,QQ)。本研究采用垫圈法和丁内酯平板法自我国不同地区采集植物的根围土壤分离具有降解AHL信号分子能力的细菌,采用16S rDNA序列分析对所分离到的群体感淬灭细菌进行初步分类鉴定。结果表明垫圈法分离得到约566株细菌菌株,其中13株具有较强降解信号分子能力,包括假单胞菌6株、不动杆菌5株、变形杆菌1株和Rheinheimeramesophila 1株。丁内酯平板法分离得到约828株细菌,其中14株具有较强降解AHL信号分子能力,包括节杆菌6株、假单胞菌6株和芽孢杆菌2株。上述结果表明,虽然垫圈法分离获得细菌总数相对较少,但比丁内酯平板法更易获得一些新型菌株。从无锡分离得到的菌株WX14降解AHL能力强,且胞内和胞外均具有降解活性,16S rDNA初步鉴定结果为荧光假单胞菌细菌。利用PCR法从菌株WX14中克隆到hacA和hacB两个群体感应淬灭酶基因,基因全长分别为2286bp和2370bp,采用DNAMAN软件分析,hacA由761个氨基酸残基组成,推测的分子量为83.7 kDa。hacB由781个氨基酸残基组成,推测的分子量为86.8 kDa。通过氨基酸序列相似性比较,HacA与已报道的Pseudomonas syringae的Psyr_1971酰基转移酶相似性可达62.74%,与Pseudomonasaeruginosa的PvdQ酰基转移酶相似性达到54.32%,HacB与已报道的Pseudomonas syringae Psyr_4858相似性达到 71.13%,与Pseudomonas aeruginosa PA0305 相似性达到 68.25%。此外hacA 与 hacB可能经历翻译后修饰形成α-和β-亚基,并且β-亚基的第一个氨基酸都是起催化作用的丝氨酸。以上结果表明hacA与hacB编码产物可能为N末端亲核(N-terminal nucleophile,Ntn)水解酶家族成员。
[Abstract]:Quorum sensing (QS) is a transduction mechanism of signaling molecules between cells of bacteria. That is, bacteria can adapt to the changeable natural environment by sensing the concentration of autoinducer AI (autoinducer AI), such as the concentration of N-acetyl homoserine lactone (AHLs) to coordinate the expression of related genes of bacteria. Many plant pathogenic bacteria regulate the expression of toxic genes by colony sensing system, so the colony sensing system is a potential target for the prevention and control of plant diseases. This interference and destruction of bacterial QS regulation mechanism is called quorum quenching QQ. In this study, bacteria with the ability to degrade AHL signaling molecules were isolated from root soil collected from different regions of China by gasket method and butyrolactone plate method. 16s rDNA sequence analysis was used to identify the isolated colony sensitive quenched bacteria. The results showed that about 566 bacterial strains were isolated by the gasket method, 13 of which had strong signal degradation ability, including 6 Pseudomonas, 5 Acinetobacter, 1 Proteus and 1 Rheinheimeramesophila. About 828 strains of bacteria were isolated by butyrolactone plate method. Among them, 14 strains had strong ability to degrade AHL signal molecules, including 6 strains of Arthrobacter, 6 strains of Pseudomonas and 2 strains of Bacillus. The results show that although the total number of bacteria isolated by gasket method is relatively small, it is easier to obtain some new strains than butyrolactone plate method. The strain WX14 isolated from Wuxi had strong ability to degrade AHL, and the biodegradable activity of 16s rDNA was identified as Pseudomonas fluorescein bacteria. The induction quenching enzyme genes of hacA and hacB were cloned from strain WX14 by PCR. The total length of the gene was 2286bp and 2370 bp.The DNA man software was used to analyze 761 amino acid residues. The deduced molecular weight was 83.7 kDa.hacB composed of 781 amino acid residues. The predicted molecular weight is 86.8 kDa. By comparison of amino acid sequence similarity, the similarity between Haca and reported Pseudomonas syringae Psyr1971 acyltransferase was 62.74, with Pseudomonas aeruginosa's PvdQ acyltransferase 54.32 HacB and Pseudomonas syringae Psyr4858 was 71.1313, and with Pseudomonas aeruginosa PA0305 was 68.25. In addition, hacA and hacB may undergo post-translational modification to form 伪-and 尾-subunits, and the first amino acids of 尾-subunits are both catalytic serine. These results suggest that hacA and hacB encodes may be members of N-terminal nucleophile Ntn hydrolase family.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476;S432.4

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