环己胺降解菌NyZ12胺氧化酶基因敲除突变体的构建及特性研究

发布时间：2018-07-31 14:50

【摘要】：本实验的目的是研究假单胞菌NyZ12降解环己胺的分子机制,找到其中起降解环己胺作用的胺氧化酶基因。环己胺是一种易燃、有毒的有机污染物,在生产和使用过程中,被释放的环己胺废气对环境和人体有很大的危害性。而微生物降解有机污染物有成本低、效率高等优点,因此找到一种能降解环己胺的微生物并研究其降解机制对降低环境中环己胺的污染至关重要。目前报道的以环己胺为碳源和氮源生长的纯培养物主要有两株,一株由日本人IwakiH.等分离筛选到的革兰氏阳性菌BrevibacteriumoxydansIH-35A,另一株由中国科学院武汉病毒研究所周宁一研究组分离的革兰氏阴性菌PseudomonasplecoglossicidaNyZ12。通过对NyZ12全基因组测序获得该菌株的全基因组信息。利用数据库比对分析,我们预测了5个基因可能与环己胺降解代谢过程有关,分别是amo425、amo2631、amo4207、amo4637和amo5539。本实验从遗传学角度研究基因是否参与环己胺代谢,对五个基因进行敲除,通过观察突变体特性变化来进一步分析基因功能。具体实验过程分为四步:1.同源片段扩增:设计合适的敲除引物扩增目的基因上下游同源臂,再利用重叠PCR技术将上下游同源臂融合。2.构建敲除载体:将融合片段克隆到pGEM-TEasy载体上转化至大肠杆菌,酶切鉴定并测序。测序鉴定正确的重组T载体与自杀载体pEX18km双酶切后连接转化到大肠杆菌并鉴定。3.结合转移:再将鉴定正确的重组质粒转化到E.coliS17λpir菌株与野生菌NyZ12进行结合转移,使含有同源片段的自杀载体在野生菌细胞内进行同源重组。4.突变体筛选及特性研究:利用抗生素抗性和PCR鉴定来筛选突变菌株,将基因敲除的突变菌株接种于环己胺无机盐培养基进行生长情况研究。设计了两种敲除方法,在目的基因内部插入抗生素基因的插入突变和利用重叠PCR技术使目的基因部分缺失的无痕敲除。结果利用插入突变敲除基因amo2631后突变体生长变慢,但考虑到抗生素基因插入可能导致的极性效应对细菌生长造成影响,后来使用无痕敲除技术进行基因敲除,结果显示单个目的基因敲除对生长没有明显影响。我们推断环己胺代谢途径中可能涉及多基因参与,后续考虑对胺氧化酶基因累积敲除。通过基因组分析发现编码分解环己醇或环己酮到己二酸的基因orf2866-2870位于一个操纵子上,转录组分析表明位于整个操纵子的五个基因受底物诱导表达水平上调。通过荧光定量PCR对操纵子上各个基因的表达水平进行进一步确认。接着对环己酮单加氧酶chnB(orf2867)进行无痕敲除,敲除突变体在环己胺和环己酮的无机盐培养基中都不生长。在突变体的restingcell中,加入环己胺,通过气相色谱在其上清液中检测到环己酮生成,证明环己胺代谢过程是通过中间产物环己酮降解的。接着对chnB敲除菌进行了回补实验,成功构建了回补突变株,其在底物环己胺和环己酮培养基上恢复生长能力。
[Abstract]:The aim of this experiment is to study the molecular mechanism of Cyclohexamide degradation by Pseudomonas NyZ12 and to find an amine oxidase gene that degrades cyclohexamine. Cyclohexamine is a flammable and toxic organic pollutant. In the process of production and use, the released cyclohexamine waste gas is very harmful to the environment and the human body. It has the advantages of low cost and high efficiency. Therefore, it is very important to find a kind of microorganism that can degrade cyclohexylamine and study its degradation mechanism to reduce the pollution of cyclohexylamine in the environment. At present, two pure cultures with cyclohexylamine as carbon source and nitrogen source are two, one is isolated and screened by Japanese. BrevibacteriumoxydansIH-35A, another strain of gram-negative PseudomonasplecoglossicidaNyZ12. isolated by the Zhou Ningyi research group of the Wuhan Institute of virus research of the Chinese Academy of Sciences, obtained the whole genome information of the strain by sequencing the whole genome of the NyZ12. Using the database comparison, we predicted that 5 genes might be associated with the ring. The metabolic process of hexamines degradation is related to amo425, amo2631, amo4207, amo4637 and amo5539.. This experiment studies whether genes participate in cyclohexamine metabolism, knock out five genes and further analyze gene function by observing the variation of mutant characteristics. The experiment process is divided into four steps: 1. homologous fragment amplification: Design Appropriate knock-out primers to amplify the target gene upstream and downstream homologous arm, and then use the overlapping PCR technique to construct the knockout vector with the upstream and downstream homologous arm fusion.2.: the fusion fragment was cloned to the pGEM-TEasy vector and converted to the Escherichia coli, and the enzyme was identified and sequenced. The correct recombinant T carrier and the suicide carrier pEX18km double enzyme digestion and transformation were identified. E. coli and identification of.3. binding transfer: then the identification of the correct recombinant plasmid was converted to E.coliS17 lambda PIR strain and wild bacteria NyZ12, and the homologous fragment was screened and characterized by the homologous recombinant.4. mutants in the wild bacteria cells: using antibiotic resistance and PCR identification to screen mutant strains. The mutant strains of gene knockout were inoculated to the inorganic salt medium of cyclohexamine for growth. Two kinds of knockout methods were designed to insert the insertion mutation of the antibiotic gene inside the target gene and the use of the overlapping PCR technique to make the deletion of the target gene partial deletion. The growth of the body is slow, but it is considered that the possible polarity effect caused by the insertion of the antibiotic gene affects the growth of the bacteria. Later, the null knockout technique is used for gene knockout. The results show that the single target gene knockout has no obvious effect on the growth. We infer that the cyclohexamine pathway may involve multiple gene participation and subsequent consideration of the amines. The gene orf2866-2870 was found on a operon by genome analysis. The transcriptional analysis showed that the five genes in the entire operon were up regulated by the substrate induced expression level. The expression of water on each gene on the operon was expressed by the fluorescence determination of PCR. Further confirmation was carried out. Then the cyclohexanone monooxygenase chnB (orf2867) was no indentation, and the knockout mutant was not grown in the inorganic salt medium of cyclohexamine and cyclohexanone. Cyclohexamine was added to the mutant restingcell, and cyclohexanone was detected by gas chromatography in its supernatant. The metabolic process of cyclohexamine was proved. It was degraded by the intermediate cyclohexanone. Then the chnB knockout bacteria were regenerated and the remedial mutant strain was successfully constructed, and its growth ability was restored on the substrate cyclohexamine and cyclohexanone medium.
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X172

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