本文選題：群體感應(yīng)淬滅 + 酰基高絲氨酸內(nèi)酯��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：群體感應(yīng)(quorum sensing,QS)是一種細(xì)菌細(xì)胞間信號(hào)分子的傳遞機(jī)制,即細(xì)菌可以通過感應(yīng)自身的自體誘導(dǎo)分子(autoinducer,AI),如N-乙酰高絲氨酸內(nèi)酯(AHLs)的濃度來協(xié)調(diào)細(xì)菌的相關(guān)基因的表達(dá)以此來適應(yīng)多變的自然環(huán)境。許多植物病原細(xì)菌通過群體感應(yīng)系統(tǒng)來調(diào)控毒性基因的表達(dá),因此群體感應(yīng)系統(tǒng)是潛在的植物病害防治靶標(biāo)。這種對細(xì)菌QS調(diào)控機(jī)制的干擾和破壞稱為群體感應(yīng)淬滅(quorum quenching,QQ)。本研究采用墊圈法和丁內(nèi)酯平板法自我國不同地區(qū)采集植物的根圍土壤分離具有降解AHL信號(hào)分子能力的細(xì)菌,采用16S rDNA序列分析對所分離到的群體感淬滅細(xì)菌進(jìn)行初步分類鑒定。結(jié)果表明墊圈法分離得到約566株細(xì)菌菌株,其中13株具有較強(qiáng)降解信號(hào)分子能力,包括假單胞菌6株、不動(dòng)桿菌5株、變形桿菌1株和Rheinheimeramesophila 1株。丁內(nèi)酯平板法分離得到約828株細(xì)菌,其中14株具有較強(qiáng)降解AHL信號(hào)分子能力,包括節(jié)桿菌6株、假單胞菌6株和芽孢桿菌2株。上述結(jié)果表明,雖然墊圈法分離獲得細(xì)菌總數(shù)相對較少,但比丁內(nèi)酯平板法更易獲得一些新型菌株。從無錫分離得到的菌株WX14降解AHL能力強(qiáng),且胞內(nèi)和胞外均具有降解活性,16S rDNA初步鑒定結(jié)果為熒光假單胞菌細(xì)菌。利用PCR法從菌株WX14中克隆到hacA和hacB兩個(gè)群體感應(yīng)淬滅酶基因,基因全長分別為2286bp和2370bp,采用DNAMAN軟件分析,hacA由761個(gè)氨基酸殘基組成,推測的分子量為83.7 kDa。hacB由781個(gè)氨基酸殘基組成,推測的分子量為86.8 kDa。通過氨基酸序列相似性比較,HacA與已報(bào)道的Pseudomonas syringae的Psyr_1971�；D(zhuǎn)移酶相似性可達(dá)62.74%,與Pseudomonasaeruginosa的PvdQ�；D(zhuǎn)移酶相似性達(dá)到54.32%,HacB與已報(bào)道的Pseudomonas syringae Psyr_4858相似性達(dá)到 71.13%,與Pseudomonas aeruginosa PA0305 相似性達(dá)到 68.25%。此外hacA 與 hacB可能經(jīng)歷翻譯后修飾形成α-和β-亞基,并且β-亞基的第一個(gè)氨基酸都是起催化作用的絲氨酸。以上結(jié)果表明hacA與hacB編碼產(chǎn)物可能為N末端親核(N-terminal nucleophile,Ntn)水解酶家族成員。
[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 偽-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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476;S432.4

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本文編號(hào)：2060738