環(huán)己胺降解菌NyZ12胺氧化酶基因敲除突變體的構(gòu)建及特性研究
發(fā)布時間：2018-07-31 14:50
【摘要】：本實驗的目的是研究假單胞菌NyZ12降解環(huán)己胺的分子機制,找到其中起降解環(huán)己胺作用的胺氧化酶基因。環(huán)己胺是一種易燃、有毒的有機污染物,在生產(chǎn)和使用過程中,被釋放的環(huán)己胺廢氣對環(huán)境和人體有很大的危害性。而微生物降解有機污染物有成本低、效率高等優(yōu)點,因此找到一種能降解環(huán)己胺的微生物并研究其降解機制對降低環(huán)境中環(huán)己胺的污染至關(guān)重要。目前報道的以環(huán)己胺為碳源和氮源生長的純培養(yǎng)物主要有兩株,一株由日本人IwakiH.等分離篩選到的革蘭氏陽性菌BrevibacteriumoxydansIH-35A,另一株由中國科學(xué)院武漢病毒研究所周寧一研究組分離的革蘭氏陰性菌PseudomonasplecoglossicidaNyZ12。通過對NyZ12全基因組測序獲得該菌株的全基因組信息。利用數(shù)據(jù)庫比對分析,我們預(yù)測了5個基因可能與環(huán)己胺降解代謝過程有關(guān),分別是amo425、amo2631、amo4207、amo4637和amo5539。本實驗從遺傳學(xué)角度研究基因是否參與環(huán)己胺代謝,對五個基因進(jìn)行敲除,通過觀察突變體特性變化來進(jìn)一步分析基因功能。具體實驗過程分為四步:1.同源片段擴增:設(shè)計合適的敲除引物擴增目的基因上下游同源臂,再利用重疊PCR技術(shù)將上下游同源臂融合。2.構(gòu)建敲除載體:將融合片段克隆到pGEM-TEasy載體上轉(zhuǎn)化至大腸桿菌,酶切鑒定并測序。測序鑒定正確的重組T載體與自殺載體pEX18km雙酶切后連接轉(zhuǎn)化到大腸桿菌并鑒定。3.結(jié)合轉(zhuǎn)移:再將鑒定正確的重組質(zhì)粒轉(zhuǎn)化到E.coliS17λpir菌株與野生菌NyZ12進(jìn)行結(jié)合轉(zhuǎn)移,使含有同源片段的自殺載體在野生菌細(xì)胞內(nèi)進(jìn)行同源重組。4.突變體篩選及特性研究:利用抗生素抗性和PCR鑒定來篩選突變菌株,將基因敲除的突變菌株接種于環(huán)己胺無機鹽培養(yǎng)基進(jìn)行生長情況研究。設(shè)計了兩種敲除方法,在目的基因內(nèi)部插入抗生素基因的插入突變和利用重疊PCR技術(shù)使目的基因部分缺失的無痕敲除。結(jié)果利用插入突變敲除基因amo2631后突變體生長變慢,但考慮到抗生素基因插入可能導(dǎo)致的極性效應(yīng)對細(xì)菌生長造成影響,后來使用無痕敲除技術(shù)進(jìn)行基因敲除,結(jié)果顯示單個目的基因敲除對生長沒有明顯影響。我們推斷環(huán)己胺代謝途徑中可能涉及多基因參與,后續(xù)考慮對胺氧化酶基因累積敲除。通過基因組分析發(fā)現(xiàn)編碼分解環(huán)己醇或環(huán)己酮到己二酸的基因orf2866-2870位于一個操縱子上,轉(zhuǎn)錄組分析表明位于整個操縱子的五個基因受底物誘導(dǎo)表達(dá)水平上調(diào)。通過熒光定量PCR對操縱子上各個基因的表達(dá)水平進(jìn)行進(jìn)一步確認(rèn)。接著對環(huán)己酮單加氧酶chnB(orf2867)進(jìn)行無痕敲除,敲除突變體在環(huán)己胺和環(huán)己酮的無機鹽培養(yǎng)基中都不生長。在突變體的restingcell中,加入環(huán)己胺,通過氣相色譜在其上清液中檢測到環(huán)己酮生成,證明環(huán)己胺代謝過程是通過中間產(chǎn)物環(huán)己酮降解的。接著對chnB敲除菌進(jìn)行了回補實驗,成功構(gòu)建了回補突變株,其在底物環(huán)己胺和環(huán)己酮培養(yǎng)基上恢復(fù)生長能力。
[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.
【學(xué)位授予單位】：武漢輕工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X172
本文編號：2155929
[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.
【學(xué)位授予單位】：武漢輕工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X172
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